Compositions and methods for pairwise sequencing

ABSTRACT

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/377,284, filed on Jul. 15, 2021, issued as U.S. Pat. No. 11,220,707on Jan. 11, 2022, which claims the benefit of and priority to U.S.Provisional Application No. 63/212,059 filed on Jun. 17, 2021, thecontents of each of which are incorporated herein by reference in theirentireties.

Throughout this application various publications, patents, and/or patentapplications are referenced. The disclosures of the publications,patents and/or patent applications are hereby incorporated by referencein their entireties into this application in order to more fullydescribe the state of the art to which this disclosure pertains.

TECHNICAL FIELD

The present disclosure provides compositions and methods that employ thecompositions for conducting pairwise sequencing and for generatingconcatemer template molecules for pairwise sequencing.

BACKGROUND OF THE INVENTION

Polynucleotide sequencing technology has applications in biomedicalresearch and healthcare settings. Improved methods of polynucleotiderequire enhanced surface chemistry, on-support polynucleotideamplification, and base calling. Currently, these elements producebarriers in existing sequencing technology that result in limits inthroughput and poor signal-to-noise ratio, and ultimately to increasedcosts associated with polynucleotide sequencing.

There exists a need for new polynucleotide sequencing methods withimproved surface chemistry, on-support amplification, and base calling.The present disclosure provides methods and compositions to improvesequencing of polynucleotides.

SUMMARY OF THE INVENTION

The present disclosure provides a method for pairwise sequencing,comprising: (a) providing a plurality of immobilized single strandednucleic acid concatemer template molecules, wherein individualconcatemer template molecules in the plurality are immobilized to afirst surface primer that is immobilized to a support, wherein theimmobilized first surface primers lack uridine, and wherein at least oneof the immobilized concatemer template molecules in the pluralitycomprises a uridine-containing concatemer template molecule having up to30% of thymidines replaced with uridine; (b) sequencing the plurality ofimmobilized concatemer template molecules with a plurality of solubleforward sequencing primers and (i) a plurality of a first sequencingpolymerase and a plurality of multivalent molecules and (ii) a pluralityof a second sequencing polymerase and a plurality of nucleotide analogs,thereby generating a plurality of extended forward sequencing primerstrands, wherein individual immobilized concatemer template moleculeshave two or more extended forward sequencing primer strands hybridizedthereon; (c) removing the plurality of extended forward sequencingprimer strands while retaining the immobilized concatemer templatemolecules, and conducting a primer extension reaction with a pluralityof soluble extension primers, a plurality of nucleotides and a pluralityof primer extension polymerases, thereby generating a plurality offorward extension strands that are hybridized to the retainedimmobilized concatemer template molecules; (d) removing the retainedimmobilized concatemer template molecules by generating abasic sites inthe immobilized concatemer template molecules at the nucleotide(s)having the scissile moiety and generating gaps at the abasic sites togenerate a plurality of gap-containing concatemer template moleculeswhile retaining the plurality of forward extension strands and retainingthe plurality of immobilized surface primers; and e) sequencing theplurality of retained forward extension strands with a plurality ofsoluble reverse sequencing primers and (i) a plurality of a firstsequencing polymerase and a plurality of multivalent molecules and (ii)a plurality of a second sequencing polymerase and a plurality ofnucleotide analogs, thereby generating a plurality of extended reversesequencing primer strands, wherein individual retained forward extensionstrands have two or more extended reverse sequencing primer strandshybridized thereon, wherein individual multivalent molecules of steps(b) and (e) comprise (1) a core; and (2) a plurality of nucleotide armswhich comprise (i) a core attachment moiety, (ii) a spacer, (iii) alinker, and (iv) a nucleotide unit, wherein the core is attached to theplurality of nucleotide arms via their core attachment moiety, whereinthe spacer is attached to the linker, and wherein the linker is attachedto the nucleotide unit.

In some embodiments, individual concatemer template molecules in theplurality are covalently joined to an immobilized first surface primer.In some embodiments, individual concatemer template molecules in theplurality are hybridized to an immobilized first surface primer.

In some embodiments, at least one of the plurality of immobilizedconcatemer template molecules lack a uridine. In some embodiments, theimmobilized concatemer template molecules comprise two or more copies ofthe sequence of interest, and two or more copies of a universal bindingsequence for a soluble amplification primer, and wherein the pluralityof soluble extension primers of step (c) comprise a plurality of solubleamplification primers that hybridize to the universal binding sequencefor the soluble amplification primer. In some embodiments, theimmobilized concatemer template molecules comprise two or more copies ofthe sequence of interest, and two or copies of a universal bindingsequence for a soluble forward sequencing primer, and wherein theplurality of soluble extension primers of step (c) comprise a pluralityof soluble forward sequencing primers that hybridize to the universalbinding sequence for the soluble forward sequencing primer.

In some embodiments, the plurality of first sequencing polymerases insteps (b) and (e) bind a concatemer template molecule, a solublesequencing primer and a multivalent molecule to form a binding complexwhich exhibits a persistence time of greater than 0.5 seconds, whereinthe nucleic acid template molecule comprises an immobilized concatemertemplate molecule or a retained forward extension strand, and whereinthe soluble sequencing primer comprises a soluble forward sequencingprimer or a soluble reverse sequencing primer.

In some embodiments, the core of the multivalent molecules comprisesstreptavidin and the core attachment moiety comprise biotin. In someembodiments, the spacer of the multivalent molecules comprises apolyethylene glycol (PEG) moiety. In some embodiments, the linker of themultivalent molecules comprises an aliphatic chain having 2-6 subunitsor an oligo ethylene glycol chain having 2-6 subunits. In someembodiments, the plurality of nucleotide arms attached to the core ofthe multivalent molecules have the same type of a nucleotide unit, andwherein the types of nucleotide unit is selected from a group consistingof dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the plurality of multivalent molecules of step (b)and (e) comprise one type of a multivalent molecule wherein eachmultivalent molecule in the plurality has the same type of nucleotideunit selected from a group consisting of dATP, dGTP, dCTP, dTTP anddUTP. In some embodiments, the plurality of multivalent molecules ofstep (b) and (e) comprise a mixture of any combination of two or moretypes of multivalent molecules each type having nucleotide unitsselected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.In some embodiments, the plurality of multivalent molecules of step (b)and (e) comprise fluorophore-labeled multivalent molecules.

In some embodiments, the nucleotide analog of step (b) comprises aremovable chain terminating moiety at the 3′ sugar group, and whereinthe nucleotide analog of step (e) comprises a removable chainterminating moiety at the 3′ sugar group, wherein the removable chainterminating moiety comprises an alkyl group, alkenyl group, alkynylgroup, allyl group, aryl group, benzyl group, azide group, azido group,O-azidomethyl group, amine group, amide group, keto group, isocyanategroup, phosphate group, thio group, disulfide group, carbonate group,urea group, or silyl group, and wherein the removable chain terminatingmoiety is cleavable with a chemical compound to generate an extendible3′OH moiety on the sugar group.

In some embodiments, the plurality of nucleotide analogs of steps (b)and (e) comprise one type of nucleotide selected from a group consistingof dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the pluralityof nucleotide analogs of steps (b) and (e) comprise a mixture of anycombination of two or more types of nucleotides selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

In some embodiments, the plurality of nucleotide analogs of steps (b)and (e) comprise at least one fluorophore-labeled nucleotide analog.

In some embodiments, the sequencing of step (b), comprises: a)contacting the plurality of a first sequencing polymerase to (i) aplurality of nucleic acid template molecules which comprise a pluralityof immobilized concatemer template molecules or a plurality of retainedforward extension strands, and (ii) the plurality of soluble sequencingprimers which comprise a plurality of soluble forward sequencing primersor a plurality of soluble reverse sequencing primers, wherein thecontacting is conducted under a condition suitable to form a pluralityof first complexed polymerases each comprising a first sequencingpolymerase bound to a nucleic acid duplex wherein the nucleic acidduplex comprises a nucleic acid template molecule hybridized to asoluble sequencing primer; b) contacting the plurality of firstcomplexed polymerases with a plurality of fluorophore-labeledmultivalent molecules to form a plurality of multivalent-complexedpolymerases, wherein the contacting is conducted under a conditionsuitable for binding complementary nucleotide units of the multivalentmolecules to at least two of the plurality of first complexedpolymerases, thereby forming a plurality of multivalent-complexedpolymerases, and the condition inhibits incorporation of thecomplementary nucleotide units into the hybridized sequencing primers ofthe plurality of multivalent-complexed polymerases; c) detecting theplurality of multivalent-complexed polymerases; and d) identifying thenucleo-base of the complementary nucleotide units that are bound to theplurality of first complexed polymerases in the plurality ofmultivalent-complexed polymerases, thereby determining the sequence ofthe nucleic acid template.

In some embodiments, the method further comprises: e) dissociating theplurality of multivalent-complexed polymerases by removing the pluralityof first sequencing polymerases and their bound multivalent molecules,and retaining the plurality of nucleic acid duplexes; f) contacting theplurality of the retained nucleic acid duplexes of step (e) with aplurality of a second sequencing polymerase, wherein the contacting isconducted under a condition suitable for binding the plurality of secondsequencing polymerases to the plurality of the retained nucleic acidduplexes, thereby forming a plurality of second complexed polymeraseseach comprising a second sequencing polymerase bound to a retainednucleic acid duplex; g) contacting the plurality of second complexedpolymerases with a plurality of nucleotides, wherein the contacting isconducted under a condition suitable for binding complementarynucleotides to at least two of the second complexed polymerases of step(f) thereby forming a plurality of nucleotide-complexed polymerases andthe condition is suitable for promoting incorporation of the boundcomplementary nucleotides into the hybridized sequencing primers of thenucleotide-complexed polymerases, thereby generating a plurality ofextended sequencing primer strands wherein the plurality of extendedsequencing primer strands comprise a plurality of extended forwardsequencing primer strands or a plurality of extended reverse sequencingprimer strands.

In some embodiments, generating the abasic sites at the uridines of theimmobilized concatemer template molecules of step (d) comprisescontacting the immobilized concatemer template molecule with uracil DNAglycosylase (UDG).

In some embodiments, generating the plurality of gap-containingconcatemer template molecules of step (d) comprises contacting theretained immobilized template molecules containing one or more abasicsites with an endonuclease IV, AP lyase (e.g., DNA-apurinic lyase orDNA-apyrimidinic lyase), FPG glycosylase/AP lyase and/or endo VIIIglycosylase/AP lyase.

In some embodiments, the support comprises at least one hydrophilicpolymer coating layer and a plurality of surface primers immobilized tothe at least one hydrophilic polymer coating layer, and wherein the atleast one hydrophilic polymer coating layer has a water contact angle ofno more than 45 degrees.

In some embodiments, the at least one hydrophilic polymer coating layercomprises a molecule selected from a group consisting of polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, and dextran. In someembodiments, the at least one hydrophilic polymer coating layercomprises polymer molecules having a molecular weight of at least 1000Daltons. In some embodiments, the at least one hydrophilic polymercoating layer comprises branched polymer molecules having 4-8 branches.

In some embodiments, the support comprises: a) a first coating layercomprising a first monolayer of hydrophilic polymer molecules tetheredto the support; b) a second coating layer comprising a second monolayerof hydrophilic polymer molecules tethered to the first monolayer; and c)a third coating layer comprising a third monolayer of hydrophilicpolymer molecules tethered to the second monolayer, and wherein thehydrophilic polymer molecules of the first layer, second layer or thirdlayer comprise branched polymer layers.

In some embodiments, the surface primers are immobilized to thehydrophilic polymer molecules of the second monolayer or thirdmonolayer, and the surface primers are distributed at a plurality ofdepths throughout the second layer or the third layer. In someembodiments, one or more of the at least one hydrophilic polymer coatinglayers comprise a plurality of surface primers at a surface density ofleast 1000/μm².

The present disclosure provides a method for pairwise sequencing,comprising: a) providing a plurality of immobilized single strandednucleic acid concatemer template molecules each comprising at least onenucleotide having a scissile moiety that can be cleaved to generate anabasic site in the concatemer template molecule, wherein individualconcatemer template molecules in the plurality are immobilized to afirst surface primer that is immobilized to a support, and wherein theimmobilized first surface primer lacks a nucleotide having a scissilemoiety; b) sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands, wherein individual immobilized concatemer templatemolecules have two or more extended forward sequencing primer strandshybridized thereon; c) retaining the plurality of immobilized concatemertemplate molecules and replacing the plurality of extended forwardsequencing primer strands with a plurality of forward extension strandsthat are hybridized to the retained immobilized single stranded nucleicacid concatemer template molecules by conducting a primer extensionreaction; d) removing the retained immobilized concatemer templatemolecules by generating abasic sites in the immobilized single strandedconcatemer template molecules at the nucleotide(s) having the scissilemoiety and generating gaps at the abasic sites to generate a pluralityof gap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized surface primers; and e)sequencing the plurality of retained forward extension strands therebygenerating a plurality of extended reverse sequencing primer strands,wherein individual retained forward extension strands have two or moreextended reverse sequencing primer strands hybridized thereon.

In some embodiments, the individual concatemer template molecules in theplurality are covalently joined to an immobilized first surface primer.In some embodiments, the individual concatemer template molecules in theplurality are hybridized to an immobilized first surface primer. In someembodiments, the individual immobilized concatemer template molecules inthe plurality comprise two or more copies of a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) two or morecopies of a universal binding sequence for a soluble forward sequencingprimer, (ii) two or more copies of a universal binding sequence for asoluble reverse sequencing primer, (iii) two or more copies of auniversal binding sequence for an immobilized first surface primer, (iv)two or more copies of a universal binding sequence for an immobilizedsecond surface primer, (v) two or more copies of a universal bindingsequence for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence for a second solubleamplification primer, (vii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, the sequencing of step (b) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (e)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized concatemer template molecules andconducting one or more sequencing reactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, at least one copy of the universalbinding sequence for the immobilized second surface primer in theindividual concatemer template molecules is hybridized to an immobilizedsecond surface primer. In some embodiments, the plurality of immobilizedsecond surface primers have 3′ OH extendible ends. In some embodiments,the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

The present disclosure also provides a method for pairwise sequencing,comprising: a) providing a support having a plurality of a first surfaceprimer immobilized thereon wherein each of the first surface primershave a 3′ extendible end and lack a nucleotide having a scissile moiety;b) generating a plurality of immobilized single stranded nucleic acidconcatemer template molecules by hybridizing a plurality ofsingle-stranded circular nucleic acid library molecules to the pluralityof immobilized first surface primers and conducting a rolling circleamplification reaction with a plurality of a strand displacingpolymerase, and a plurality of nucleotides which include dATP, dCTP,dGTP, dTTP and a nucleotide having a scissile moiety that can be cleavedto generate an abasic site, thereby generating a plurality ofimmobilized single stranded nucleic acid concatemer template moleculeshaving at least one nucleotide with a scissile moiety, whereinindividual single stranded nucleic acid concatemer template moleculesare covalently joined to an immobilized first surface primer, c)sequencing the plurality of immobilized concatemer template moleculesthereby generating a plurality of extended forward sequencing primerstrands, wherein individual immobilized concatemer template moleculeshave two or more extended forward sequencing primer strands hybridizedthereon; d) retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized single stranded nucleic acidconcatemer template molecules by conducting a primer extension reaction;e) removing the retained immobilized concatemer template molecules bygenerating abasic sites in the immobilized single stranded concatemertemplate molecules at the nucleotide(s) having the scissile moiety andgenerating gaps at the abasic sites to generate a plurality ofgap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized first surface primers; and f)sequencing the plurality of retained forward extension strands therebygenerating a plurality of extended reverse sequencing primer strands,wherein individual forward extension strands have two or more extendedreverse sequencing primer strands hybridized thereon.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence for asoluble forward sequencing primer, (ii) a universal binding sequence fora soluble reverse sequencing primer, (iii) a universal binding sequencefor an immobilized first surface primer, (iv) a universal bindingsequence for an immobilized second surface primer, (v) a universalbinding sequence for a first soluble amplification primer, (vi) auniversal binding sequence for a second soluble amplification primer,(vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, the individual immobilized single stranded nucleicacid concatemer template molecules generated by the rolling circleamplification reaction comprise two or more copies of a sequence ofinterest and wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for an immobilized firstsurface primer, (iv) two or more copies of a universal binding sequencefor an immobilized second surface primer, (v) two or more copies of auniversal binding sequence for a first soluble amplification primer,(vi) two or more copies of a universal binding sequence for a secondsoluble amplification primer, (vii) two or more copies of a universalbinding sequence for a soluble compaction oligonucleotide, (viii) two ormore copies of a sample barcode sequence and/or (ix) two or more copiesof a unique molecular index sequence.

In some embodiments, the sequencing of step (c) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (f)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized concatemer template molecules andconducting one or more sequencing reactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, at least one copy of the universalbinding sequence for the immobilized second surface primer in theindividual concatemer template molecules is hybridized to an immobilizedsecond surface primer. In some embodiments, the plurality of immobilizedsecond surface primers have 3′ OH extendible ends. In some embodiments,the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

The present disclosure also provides a method for pairwise sequencing,comprising: a) contacting in-solution a plurality of single-strandedcircular nucleic acid library molecules to a plurality of first solubleamplification primers, a plurality of a strand displacing polymerase,and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP anda nucleotide having a scissile moiety that can be cleaved to generate anabasic site, under a condition suitable to form a plurality oflibrary-primer duplexes and suitable for conducting a rolling circleamplification reaction, thereby generating a plurality of singlestranded nucleic acid concatemers having at least one nucleotide with ascissile moiety; b) distributing the rolling circle amplificationreaction onto a support having a plurality of the first surface primersimmobilized thereon, under a condition suitable for hybridizing one ormore portions of individual single stranded concatemers to one or moreimmobilized first surface primers, wherein each of the first surfaceprimers lack a nucleotide having a scissile moiety; c) continuing therolling circle amplification reaction on the support to generate aplurality of immobilized concatemer template molecules; d) sequencingthe plurality of immobilized concatemer template molecules therebygenerating a plurality of extended forward sequencing primer strandswherein individual immobilized concatemer template molecules have two ormore extended forward sequencing primer strands hybridized thereon; e)retaining the plurality of immobilized concatemer template molecules andreplacing the plurality of extended forward sequencing primer strandswith a plurality of forward extension strands that are hybridized to theretained immobilized single stranded nucleic acid concatemer templatemolecules by conducting a primer extension reaction; f) removing theretained immobilized concatemer template molecules by generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotide(s) having the scissile moiety and generating gaps atthe abasic sites to generate a plurality of gap-containing singlestranded nucleic acid concatemer template molecules while retaining theplurality of forward extension strands and retaining the plurality ofimmobilized first surface primers; and g) sequencing the plurality ofretained forward extension strands thereby generating a plurality ofextended reverse sequencing primer strands wherein individual forwardextension strands have two or more extended reverse sequencing primerstrands hybridized thereon.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence for asoluble forward sequencing primer, (ii) a universal binding sequence fora soluble reverse sequencing primer, (iii) a universal binding sequencefor an immobilized first surface primer, (iv) a universal bindingsequence for an immobilized second surface primer, (v) a universalbinding sequence for a first soluble amplification primer, (vi) auniversal binding sequence for a second soluble amplification primer,(vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecules generated by the rolling circleamplification reaction comprise two or more copies of a sequence ofinterest and wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for an immobilized firstsurface primer, (iv) two or more copies of a universal binding sequencefor an immobilized second surface primer, (v) two or more copies of auniversal binding sequence for a first soluble amplification primer,(vi) two or more copies of a universal binding sequence for a secondsoluble amplification primer, (vii) two or more copies of a universalbinding sequence for a soluble compaction oligonucleotide, (viii) two ormore copies of a sample barcode sequence and/or (ix) two or more copiesof a unique molecular index sequence.

In some embodiments, the sequencing of step (d) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (g)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized concatemer template molecules andconducting one or more sequencing reactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, at least one copy of the universalbinding sequence for the immobilized second surface primer in theindividual concatemer template molecules is hybridized to an immobilizedsecond surface primer. In some embodiments, the plurality of immobilizedsecond surface primers have 3′ OH extendible ends. In some embodiments,the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

The present disclosure provides a method for pairwise sequencing,comprising: a) providing a support having a plurality of a first surfaceprimer immobilized thereon wherein individual first surface primers inthe plurality comprise a first portion (SP1-A) and a second portion(SP1-B), and the individual first surface primers comprising a 3′extendible end and lacking a nucleotide having a scissile moiety thatcan be cleaved to generate an abasic site in the first surface primer;b) contacting the plurality of the first surface primers with aplurality of single stranded linear nucleic acid library molecules, eachlibrary molecule having at the 5′ end a universal sequence (SP1-A′) thatbinds the first portion of the immobilized first surface primer, and thelibrary molecules each having at the 3′ end a universal sequence(SP1-B′) that binds the second portion of the immobilized first surfaceprimer, wherein the contacting is conducted under a condition suitablefor hybridizing individual library molecules to an immobilized firstsurface primer to form a circularized library molecule having a gap ornick between the 5′ and 3′ ends of the circularized library molecule; c)enzymatically closing the gap or nick thereby forming individualcovalently closed circular molecules that are hybridized to animmobilized first surface primer; d) generating a plurality ofimmobilized single stranded nucleic acid concatemer template moleculesby conducting a rolling circle amplification reaction with a pluralityof a strand displacing polymerase, and a plurality of nucleotides whichinclude dATP, dCTP, dGTP, dTTP and a nucleotide having a scissile moietythat can be cleaved to generate an abasic site, thereby generating aplurality of immobilized single stranded nucleic acid concatemertemplate molecules having at least one nucleotide with a scissilemoiety, wherein individual single stranded nucleic acid concatemertemplate molecules are covalently joined to an immobilized first surfaceprimer; e) sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands, wherein individual immobilized concatemer templatemolecules have two or more extended forward sequencing primer strandshybridized thereon; f) retaining the plurality of immobilized concatemertemplate molecules and replacing the plurality of extended forwardsequencing primer strands with a plurality of forward extension strandsthat are hybridized to the retained immobilized single stranded nucleicacid concatemer template molecules by conducting a primer extensionreaction; g) removing the retained immobilized concatemer templatemolecules by generating abasic sites in the immobilized single strandedconcatemer template molecules at the nucleotide(s) having the scissilemoiety and generating gaps at the abasic sites to generate a pluralityof gap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized first surface primers; and h)sequencing the plurality of retained forward extension strands therebygenerating a plurality of extended reverse sequencing primer strands,wherein individual forward extension strands have two or more extendedreverse sequencing primer strands hybridized thereon.

In some embodiments, individual linear library molecules in theplurality comprise a sequence of interest and the library moleculesfurther comprise any one or any combination of two or more of: (i) auniversal binding sequence for a soluble forward sequencing primer, (ii)a universal binding sequence for a soluble reverse sequencing primer,(iii) a universal binding sequence for a first portion of an immobilizedfirst surface primer (SP1-A), (iv) a universal binding sequence for asecond portion of an immobilized first surface primer (SP1-B), (v) auniversal binding sequence for an immobilized second surface primer,(vi) a universal binding sequence for a first soluble amplificationprimer, (vii) a universal binding sequence for a second solubleamplification primer, (viii) a universal binding sequence for a solublecompaction oligonucleotide, (ix) a sample barcode sequence and/or (x) aunique molecular index sequence.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecules generated by the rolling circleamplification reaction comprise two or more copies of a sequence ofinterest and wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for a first portion of animmobilized first surface primer (SP1-A), (iv) two or more copies of auniversal binding sequence for a second portion of an immobilized firstsurface primer (SP1-B), (v) two or more copies of a universal bindingsequence for an immobilized second surface primer, (vi) two or morecopies of a universal binding sequence for a first soluble amplificationprimer, (vii) two or more copies of a universal binding sequence for asecond soluble amplification primer, (viii) two or more copies of auniversal binding sequence for a soluble compaction oligonucleotide,(ix) two or more copies of a sample barcode sequence and/or (x) two ormore copies of a unique molecular index sequence.

In some embodiments, the sequencing of step (e) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (h)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized concatemer template molecules andconducting one or more sequencing reactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, at least one copy of the universalbinding sequence for the immobilized second surface primer in theindividual concatemer template molecules is hybridized to an immobilizedsecond surface primer. In some embodiments, the plurality of immobilizedsecond surface primers have 3′ OH extendible ends. In some embodiments,the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

In some embodiments, the closing the gap in the circularized librarymolecule comprises conducting a polymerase-catalyzed gap fill-inreaction using the immobilized first surface primer as a templatemolecule, and ligating the nick to form a covalently closed circularmolecule, wherein individual covalently closed circular molecules arehybridized to an immobilized first surface primer. In some embodiments,the closing the nick in the circularized library molecule comprisesconducting a ligation reaction to form a covalently closed circularmolecule, and wherein individual covalently closed circular moleculesare hybridized to an immobilized first surface primer.

The present disclosure provides a method for pairwise sequencing,comprising: a) providing a plurality of immobilized single strandednucleic acid concatemer template molecules each lacking a scissilemoiety that can be cleaved to generate an abasic site in the concatemertemplate molecule, wherein individual concatemer template molecules inthe plurality are immobilized to a first surface primer that isimmobilized to a support, and wherein the immobilized first surfaceprimer lacks a nucleotide having a scissile moiety; b) sequencing theplurality of immobilized concatemer template molecules therebygenerating a plurality of extended forward sequencing primer strands,wherein individual immobilized concatemer template molecules have two ormore extended forward sequencing primer strands hybridized thereon; c)retaining the plurality of immobilized concatemer template molecules andreplacing the plurality of extended forward sequencing primer strandswith a plurality of forward extension strands by conducting a primerextension reaction with a plurality of soluble amplification primers anda plurality of strand-displacing polymerases to generate a plurality offorward extension strands and a plurality of partially displaced forwardextension strands that are hybridized to the immobilized concatemertemplate molecules to form a plurality of immobilized amplicons, and theprimer extension reaction generates a plurality of detached forwardextension strands (e.g., that are not hybridized to the immobilizedconcatemer template molecules); and d) sequencing the plurality ofimmobilized partially displaced forward extension strands therebygenerating a first plurality of extended reverse sequencing primerstrands, and sequencing the plurality of immobilized detached forwardextension strands thereby generating a second plurality of extendedreverse sequencing primer strands, wherein individual immobilizedpartially displaced forward extension strands have two or more extendedreverse sequencing primer strands hybridized thereon, and wherein inindividual immobilized detached forward extension strands have two ormore extended reverse sequencing primer strands hybridized thereon.

In some embodiments, individual concatemer template molecules in theplurality are covalently joined to an immobilized first surface primer.In some embodiments, individual concatemer template molecules in theplurality are hybridized to an immobilized first surface primer. In someembodiments, individual immobilized concatemer template molecules in theplurality comprise two or more copies of a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) two or morecopies of a universal binding sequence for a soluble forward sequencingprimer, (ii) two or more copies of a universal binding sequence for asoluble reverse sequencing primer, (iii) two or more copies of auniversal binding sequence for an immobilized first surface primer, (iv)two or more copies of a universal binding sequence for an immobilizedsecond surface primer, (v) two or more copies of a universal bindingsequence for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence for a second solubleamplification primer, (vii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, the sequencing of step (b) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (d)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized partially displaced forward extensionstrands and the plurality of immobilized detached extended forwardsequencing primer strands, and conducting one or more sequencingreactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, at least one copy of the universalbinding sequence for the immobilized second surface primer in theindividual concatemer template molecules is hybridized to an immobilizedsecond surface primer. In some embodiments, the plurality of immobilizedsecond surface primers have 3′ OH extendible ends. In some embodiments,the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

The present disclosure also provides a method for pairwise sequencing,comprising: a) providing a support having a plurality of a first surfaceprimer immobilized thereon wherein each of the first surface primershave a 3′ extendible end and lack a nucleotide having a scissile moiety;b) generating a plurality of immobilized single stranded nucleic acidconcatemer template molecules by hybridizing a plurality ofsingle-stranded circular nucleic acid library molecules to the pluralityof immobilized first surface primers and conducting a rolling circleamplification reaction with a plurality of a strand displacingpolymerase, and a plurality of nucleotides which lack a nucleotidehaving a scissile moiety that can be cleaved to generate an abasic site,thereby generating a plurality of immobilized single stranded nucleicacid concatemer template molecules, wherein individual single strandednucleic acid concatemer template molecules are covalently joined to animmobilized first surface primer; c) sequencing the plurality ofimmobilized concatemer template molecules thereby generating a pluralityof extended forward sequencing primer strands, wherein individualimmobilized concatemer template molecules have two or more extendedforward sequencing primer strands hybridized thereon; d) retaining theplurality of immobilized concatemer template molecules and replacing theplurality of extended forward sequencing primer strands with a pluralityof forward extension strands by conducting a primer extension reactionwith a plurality of soluble amplification primers and a plurality ofstrand-displacing polymerases to generate a plurality of forwardextension strands and a plurality of partially displaced forwardextension strands that are hybridized to the immobilized concatemertemplate molecules to form a plurality of immobilized amplicons, and theprimer extension reaction generates a plurality of detached forwardextension strands (e.g., that are not hybridized to the immobilizedconcatemer template molecules); and e) sequencing the plurality ofimmobilized partially displaced forward extension strands therebygenerating a first plurality of extended reverse sequencing primerstrands, and sequencing the plurality of immobilized detached forwardextension strands thereby generating a second plurality of extendedreverse sequencing primer strands, wherein individual immobilizedpartially displaced forward extension strands have two or more extendedreverse sequencing primer strands hybridized thereon, and wherein inindividual immobilized detached forward extension strands have two ormore extended reverse sequencing primer strands hybridized thereon.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest, andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence for asoluble forward sequencing primer, (ii) a universal binding sequence fora soluble reverse sequencing primer, (iii) a universal binding sequencefor an immobilized first surface primer, (iv) a universal bindingsequence for an immobilized second surface primer, (v) a universalbinding sequence for a first soluble amplification primer, (vi) auniversal binding sequence for a second soluble amplification primer,(vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecules generated by the rolling circleamplification reaction comprise two or more copies of a sequence ofinterest, wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for an immobilized firstsurface primer, (iv) two or more copies of a universal binding sequencefor an immobilized second surface primer, (v) two or more copies of auniversal binding sequence for a first soluble amplification primer,(vi) two or more copies of a universal binding sequence for a secondsoluble amplification primer, (vii) two or more copies of a universalbinding sequence for a soluble compaction oligonucleotide, (viii) two ormore copies of a sample barcode sequence and/or (ix) two or more copiesof a unique molecular index sequence.

In some embodiments, the sequencing of step (c) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (e)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized partially displaced forward extensionstrands and the plurality of immobilized detached extended forwardsequencing primer strands, and conducting one or more sequencingreactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, the at least one copy of theuniversal binding sequence for the immobilized second surface primer inthe individual concatemer template molecules is hybridized to animmobilized second surface primer. In some embodiments, the plurality ofimmobilized second surface primers have 3′ OH extendible ends. In someembodiments, the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

The present disclosure also provides a method for pairwise sequencing,comprising: a) contacting in-solution a plurality of single-strandedcircular nucleic acid library molecules to a plurality of first solubleamplification primers, a plurality of a strand displacing polymerase,and a plurality of nucleotides which lacks a nucleotide having ascissile moiety that can be cleaved to generate an abasic site, under acondition suitable to form a plurality of library-primer duplexes andsuitable for conducting a rolling circle amplification reaction, therebygenerating a plurality of single stranded nucleic acid concatemers; b)distributing the rolling circle amplification reaction onto a supporthaving a plurality of the first surface primers immobilized thereon,under a condition suitable for hybridizing one or more portions ofindividual single stranded concatemers to one or more immobilized firstsurface primers, wherein each of the first surface primers lack anucleotide having a scissile moiety; c) continuing the rolling circleamplification reaction on the support to generate a plurality ofimmobilized concatemer template molecules; d) sequencing the pluralityof immobilized concatemer template molecules thereby generating aplurality of extended forward sequencing primer strands whereinindividual immobilized concatemer template molecules have two or moreextended forward sequencing primer strands hybridized thereon; e)retaining the plurality of immobilized concatemer template molecules andreplacing the plurality of extended forward sequencing primer strandswith a plurality of forward extension strands by conducting a primerextension reaction with a plurality of a second soluble amplificationprimer and a plurality of strand-displacing polymerases to generate aplurality of forward extension strands and a plurality of partiallydisplaced forward extension strands that are hybridized to theimmobilized concatemer template molecules to form a plurality ofimmobilized amplicons, and the primer extension reaction generates aplurality of detached forward extension strands (e.g., that are nothybridized to the immobilized concatemer template molecules); and f)sequencing the plurality of immobilized partially displaced forwardextension strands thereby generating a first plurality of extendedreverse sequencing primer strands, and sequencing the plurality ofimmobilized detached forward extension strands thereby generating asecond plurality of extended reverse sequencing primer strands, whereinindividual immobilized partially displaced forward extension strandshave two or more extended reverse sequencing primer strands hybridizedthereon, and wherein in individual immobilized detached forwardextension strands have two or more extended reverse sequencing primerstrands hybridized thereon.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest, andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence for asoluble forward sequencing primer, (ii) a universal binding sequence fora soluble reverse sequencing primer, (iii) a universal binding sequencefor an immobilized first surface primer, (iv) a universal bindingsequence for an immobilized second surface primer, (v) a universalbinding sequence for a first soluble amplification primer, (vi) auniversal binding sequence for a second soluble amplification primer,(vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecules generated by the rolling circleamplification reaction comprise two or more copies of a sequence ofinterest, and wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for an immobilized firstsurface primer, (iv) two or more copies of a universal binding sequencefor an immobilized second surface primer, (v) two or more copies of auniversal binding sequence for a first soluble amplification primer,(vi) two or more copies of a universal binding sequence for a secondsoluble amplification primer, (vii) two or more copies of a universalbinding sequence for a soluble compaction oligonucleotide, (viii) two ormore copies of a sample barcode sequence and/or (ix) two or more copiesof a unique molecular index sequence.

In some embodiments, the sequencing of step (d) comprises hybridizing aplurality of soluble forward sequencing primers to the plurality ofimmobilized concatemer template molecules and conducting one or moresequencing reactions. In some embodiments, the sequencing of step (f)comprises hybridizing a plurality of soluble reverse sequencing primersto the plurality of immobilized partially displaced forward extensionstrands and the plurality of immobilized detached extended forwardsequencing primer strands, and conducting one or more sequencingreactions.

In some embodiments, the support further comprises a plurality ofimmobilized second surface primers that lack a nucleotide having ascissile moiety. In some embodiments, at least one copy of the universalbinding sequence for the immobilized second surface primer in theindividual concatemer template molecules is hybridized to an immobilizedsecond surface primer. In some embodiments, the plurality of immobilizedsecond surface primers have 3′ OH extendible ends. In some embodiments,the plurality of immobilized second surface primers have 3′non-extendible ends. In some embodiments, the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.

The present disclosure also provides a method for pairwise sequencing,comprising: a) providing a plurality of immobilized single strandednucleic acid concatemer template molecules each comprising at least onenucleotide having a scissile moiety that can be cleaved to generate anabasic site in the concatemer template molecule, wherein individualconcatemer template molecules in the plurality are immobilized to afirst surface primer that is immobilized to a support, wherein theimmobilized first surface primers include a nucleotide having a scissilemoiety, wherein the support further comprises a plurality of immobilizedsecond surface primers which lack a nucleotide having a scissile moietyand have an extendible terminal 3′OH group, and wherein the immobilizedconcatemer template molecule comprises two or more copies of a universalbinding sequence for an immobilized second surface primer (wherein thesupport comprises an excess of immobilized first and second surfaceprimers compared to the number of immobilized concatemer templatemolecules); b) sequencing the plurality of immobilized concatemertemplate molecules with a plurality of soluble forward sequencingprimers thereby generating a plurality of extended forward sequencingprimer strands, wherein individual immobilized concatemer templatemolecules have two or more extended forward sequencing primer strandshybridized thereon; c) removing the extended forward sequencing primerstrands and retaining the immobilized concatemer template molecules; d)generating a first plurality of immobilized forward extension strands byhybridizing at least one portion of individual immobilized concatemertemplate molecules to a second surface primer and conducting a primerextension reaction from the second surface primers that are hybridizedto a portion of the immobilized concatemer template molecule to generatea plurality of forward extension strands having a sequence that iscomplementary to at least a portion of the immobilized concatemertemplate molecules and are covalently joined to an immobilized secondsurface primer; e) contacting the plurality of immobilized concatemertemplate molecules and the plurality of immobilized forward extensionstrands with a relaxing solution which comprises at least one chaotropicagent; f) dissociating the at least one portion of the immobilizedconcatemer template molecules from the immobilized second surfaceprimers and retaining the immobilized forward extension strands, andre-hybridizing at least one portion of the immobilized concatemertemplate molecules to one of the immobilized second surface primers thatare not covalently joined to a forward extension strand, wherein thedissociating and re-associating comprises a temperature ramp-up, atemperature plateau, and temperature ramp-down, and washing the relaxingsolution from the support; g) contacting the re-hybridized immobilizedconcatemer template molecules with an amplification solution andconducting a primer extension reaction from the second surface primersthat are re-hybridized to a portion of the immobilized concatemertemplate molecules to generate a plurality of newly synthesized forwardextension strands having a sequence that is complementary to at least aportion of the immobilized concatemer template molecules and arecovalently joined to an immobilized second surface primer; h) repeatingsteps (e)-(g) at least once; i) removing the retained immobilizedconcatemer template molecules by generating abasic sites in theimmobilized single stranded concatemer template molecules and theimmobilized first surface primers at the nucleotide(s) having thescissile moiety and generating gaps at the abasic sites therebygenerating a plurality of gap-containing nucleic acid molecules whileretaining the plurality of immobilized forward extension strands andretaining the plurality of immobilized second surface primers; and j)sequencing the plurality of retained immobilized forward extensionstrands with a plurality of soluble reverse sequencing primers therebygenerating a plurality of extended reverse sequencing primer strands.

In some embodiments, individual concatemer template molecules in theplurality are covalently joined to an immobilized first surface primer.In some embodiments, individual concatemer template molecules in theplurality are hybridized to an immobilized first surface primer. In someembodiments, individual immobilized concatemer template molecules in theplurality comprise two or more copies of a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) two or morecopies of a universal binding sequence for a soluble forward sequencingprimer, (ii) two or more copies of a universal binding sequence for asoluble reverse sequencing primer, (iii) two or more copies of auniversal binding sequence for an immobilized first surface primer, (iv)two or more copies of a universal binding sequence for an immobilizedsecond surface primer, (v) two or more copies of a universal bindingsequence for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence for a second solubleamplification primer, (vii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

The present disclosure also provides a method for pairwise sequencing,comprising: a) providing a support having a plurality of first andsecond surface primers immobilized thereon, wherein the first surfaceprimers have a scissile moiety that can be cleaved to generate an abasicsite, and wherein the second surface primers lack a nucleotide having ascissile moiety and the second surface primers have an extendibleterminal 3′OH group; b) generating a plurality of immobilized singlestranded nucleic acid concatemer template molecules by hybridizing aplurality of single-stranded circular nucleic acid library molecules tothe plurality of immobilized first surface primers and conducting arolling circle amplification reaction with a plurality of a stranddisplacing polymerase, and a plurality of nucleotides which includedATP, dCTP, dGTP, dTTP and a plurality of nucleotides having a scissilemoiety that can be cleaved to generate an abasic site, therebygenerating a plurality of immobilized single stranded nucleic acidconcatemer template molecules having at least one nucleotide with ascissile moiety, wherein individual single stranded nucleic acidconcatemer template molecules are covalently joined to an immobilizedfirst surface primer; c) sequencing the plurality of immobilizedconcatemer template molecules with a plurality of soluble forwardsequencing primers thereby generating a plurality of extended forwardsequencing primer strands, wherein individual immobilized concatemertemplate molecules have two or more extended forward sequencing primerstrands hybridized thereon; d) removing the extended forward sequencingprimer strands and retaining the immobilized concatemer templatemolecules; e) generating a first plurality of immobilized forwardextension strands by hybridizing at least one portion of individualimmobilized concatemer template molecules to a second surface primer andconducting a primer extension reaction from the second surface primersthat are hybridized to a portion of the immobilized concatemer templatemolecule to generate a plurality of forward extension strands having asequence that is complementary to at least a portion of the immobilizedconcatemer template molecules and are covalently joined to animmobilized second surface primer; f) contacting the plurality ofimmobilized concatemer template molecules and the plurality ofimmobilized forward extension strands with a relaxing solution whichcomprises at least one chaotropic agent; g) dissociating the at leastone portion of the immobilized concatemer template molecules from theimmobilized second surface primers and retaining the immobilized forwardextension strands, and re-hybridizing at least one portion of theimmobilized concatemer template molecules to one of the immobilizedsecond surface primers that are not covalently joined to a forwardextension strand, wherein the dissociating and re-associating comprisesa temperature ramp-up, a temperature plateau, and temperature ramp-down,and washing the relaxing solution from the support; h) contacting there-hybridized immobilized concatemer template molecules with anamplification solution and conducting a primer extension reaction fromthe second surface primers that are re-hybridized to a portion of theimmobilized concatemer template molecules to generate a plurality ofnewly synthesized forward extension strands having a sequence that iscomplementary to at least a portion of the immobilized concatemertemplate molecules and are covalently joined to an immobilized secondsurface primer; i) repeating steps (f)-(h) at least once; j) removingthe retained immobilized concatemer template molecules by generatingabasic sites in the immobilized single stranded concatemer templatemolecules and the immobilized first surface primers at the nucleotide(s)having the scissile moiety and generating gaps at the abasic sites togenerate a plurality of gap-containing nucleic acid molecules whileretaining the plurality of immobilized forward extension strands andretaining the plurality of immobilized second surface primers; and k)sequencing the plurality of retained immobilized forward extensionstrands with a plurality of soluble reverse sequencing primers therebygenerating a plurality of extended reverse sequencing primer strands.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence for asoluble forward sequencing primer, (ii) a universal binding sequence fora soluble reverse sequencing primer, (iii) a universal binding sequencefor an immobilized first surface primer, (iv) a universal bindingsequence for an immobilized second surface primer, (v) a universalbinding sequence for a first soluble amplification primer, (vi) auniversal binding sequence for a second soluble amplification primer,(vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual immobilized concatemer templatemolecules in the plurality comprise two or more copies of a sequence ofinterest, and wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for an immobilized firstsurface primer, (iv) two or more copies of a universal binding sequencefor an immobilized second surface primer, (v) two or more copies of auniversal binding sequence for a first soluble amplification primer,(vi) two or more copies of a universal binding sequence for a secondsoluble amplification primer, (vii) two or more copies of a universalbinding sequence for a soluble compaction oligonucleotide, (viii) two ormore copies of a sample barcode sequence and/or (ix) two or more copiesof a unique molecular index sequence.

The present disclosure also provides a method for pairwise sequencing,comprising: a) contacting in-solution a plurality of single-strandedcircular nucleic acid library molecules to a plurality of first solubleamplification primers, a plurality of a strand displacing polymerase,and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP anda plurality of nucleotides having a scissile moiety that can be cleavedto generate an abasic site, under a condition suitable to form aplurality of library-primer duplexes and suitable for conducting arolling circle amplification reaction, thereby generating a plurality ofsingle stranded nucleic acid concatemers having at least one nucleotidewith a scissile moiety; b) distributing the rolling circle amplificationreaction onto a support having a plurality of the first surface primersimmobilized thereon, under a condition suitable for hybridizing one ormore portions of individual single stranded concatemers to one or moreimmobilized first surface primers, wherein the immobilized first surfaceprimers include a nucleotide having a scissile moiety, wherein thesupport further comprises a plurality of immobilized second surfaceprimers which lack a nucleotide having a scissile moiety and have anextendible terminal 3′OH group; c) continuing the rolling circleamplification reaction on the support in the presence of a plurality ofnucleotides which include a plurality of nucleotides having a scissilemoiety to generate a plurality of immobilized concatemer templatemolecules; d) sequencing the plurality of immobilized concatemertemplate molecules with a plurality of soluble forward sequencingprimers thereby generating a plurality of extended forward sequencingprimer strands, wherein individual immobilized concatemer templatemolecules have two or more extended forward sequencing primer strandshybridized thereon; e) removing the extended forward sequencing primerstrands and retaining the immobilized concatemer template molecules; f)generating a first plurality of immobilized forward extension strands byhybridizing at least one portion of individual immobilized concatemertemplate molecules to a second surface primer and conducting a primerextension reaction from the second surface primers that are hybridizedto a portion of the immobilized concatemer template molecule to generatea plurality of forward extension strands having a sequence that iscomplementary to at least a portion of the immobilized concatemertemplate molecules and are covalently joined to an immobilized secondsurface primer; g) contacting the plurality of immobilized concatemertemplate molecules and the plurality of immobilized forward extensionstrands with a relaxing solution which comprises at least one chaotropicagent; h) dissociating the at least one portion of the immobilizedconcatemer template molecules from the immobilized second surfaceprimers and retaining the immobilized forward extension strands, andre-hybridizing at least one portion of the immobilized concatemertemplate molecules to one of the immobilized second surface primers thatare not covalently joined to a forward extension strand, wherein thedissociating and re-associating comprises a temperature ramp-up, atemperature plateau, and temperature ramp-down, and washing the relaxingsolution from the support; i) contacting the re-hybridized immobilizedconcatemer template molecules with an amplification solution andconducting a primer extension reaction from the second surface primersthat are re-hybridized to a portion of the immobilized concatemertemplate molecules to generate a plurality of newly synthesized forwardextension strands having a sequence that is complementary to at least aportion of the immobilized concatemer template molecules and arecovalently joined to an immobilized second surface primer; j) repeatingsteps (g)-(i) at least once; k) removing the retained immobilizedconcatemer template molecules by generating abasic sites in theimmobilized single stranded concatemer template molecules and theimmobilized first surface primers at the nucleotide(s) having thescissile moiety and generating gaps at the abasic sites to generate aplurality of gap-containing nucleic acid molecules while retaining theplurality of immobilized forward extension strands and retaining theplurality of immobilized second surface primers; and l) sequencing theplurality of retained immobilized forward extension strands with aplurality of soluble reverse sequencing primers thereby generating aplurality of extended reverse sequencing primer strands.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence for asoluble forward sequencing primer, (ii) a universal binding sequence fora soluble reverse sequencing primer, (iii) a universal binding sequencefor an immobilized first surface primer, (iv) a universal bindingsequence for an immobilized second surface primer, (v) a universalbinding sequence for a first soluble amplification primer, (vi) auniversal binding sequence for a second soluble amplification primer,(vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual immobilized concatemer templatemolecules in the plurality comprise two or more copies of a sequence ofinterest, and wherein the individual immobilized concatemer templatemolecules further comprise any one or any combination of two or more of(i) two or more copies of a universal binding sequence for a solubleforward sequencing primer, (ii) two or more copies of a universalbinding sequence for a soluble reverse sequencing primer, (iii) two ormore copies of a universal binding sequence for an immobilized firstsurface primer, (iv) two or more copies of a universal binding sequencefor an immobilized second surface primer, (v) two or more copies of auniversal binding sequence for a first soluble amplification primer,(vi) two or more copies of a universal binding sequence for a secondsoluble amplification primer, (vii) two or more copies of a universalbinding sequence for a soluble compaction oligonucleotide, (viii) two ormore copies of a sample barcode sequence and/or (ix) two or more copiesof a unique molecular index sequence.

In any of the foregoing or related embodiments, the support comprises aplanar substrate which comprises glass, fused-silica, silicon, a polymer(e.g., polystyrene (PS), macroporous polystyrene (MPPS),polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP),polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET)), or any combination thereof.

In any of the foregoing or related embodiments, the support comprises atleast one hydrophilic polymer coating having a water contact angle of nomore than 45 degrees, and wherein at least one of the hydrophilicpolymer coatings comprising branched hydrophilic polymer having at least4 branches.

In any of the foregoing or related embodiments, the 5′ end of theplurality of first surface primers are immobilized to the support orimmobilized to a coating on the support. In any of the foregoing orrelated embodiments, the plurality of first surface primers comprisemodified oligonucleotide molecules having 2-10 phosphorothioate linkagesat their 5′ ends to confer resistance to nuclease degradation.

In any of the foregoing or related embodiments, the 5′ end of theplurality of second surface primers are immobilized to the support orimmobilized to a coating on the support. In some embodiments, theplurality of second surface primers comprise modified oligonucleotidemolecules having 2-10 phosphorothioate linkages at their 5′ ends toconfer resistance to nuclease degradation.

In any of the foregoing or related embodiments, the immobilizedconcatemer template molecules comprise at least one nucleotide having ascissile moiety which comprises uridine, 8-oxo-7,8-dihydrogunine, ordeoxyinosine.

In any of the foregoing or related embodiments, the nucleotides with ascissile moiety are located at randomly distributed positions inindividual immobilized concatemer template molecules in the plurality.

In any of the foregoing or related embodiments, 0.01-30% of thethymidine nucleotides in the individual immobilized concatemer templatemolecules are replaced with uridine. In any of the foregoing or relatedembodiments, 0.01-30% of the guanosine nucleotides in the individualimmobilized concatemer template molecules are replaced with8-oxo-7,8-dihydrogunine or deoxyinosine.

In any of the foregoing or related embodiments, the soluble forwardsequencing primer comprises a 3′ OH extendible end and lacks anucleotide having a scissile moiety. In any of the foregoing or relatedembodiments, the soluble reverse sequencing primer comprises a 3′ OHextendible end and lacks a nucleotide having a scissile moiety.

In any of the foregoing or related embodiments, the first solubleamplification primer comprises a 3′ OH extendible end and lacks anucleotide having a scissile moiety. In any of the foregoing or relatedembodiments, the second soluble amplification primer comprises a 3′ OHextendible end and lacks a nucleotide having a scissile moiety.

In any of the foregoing or related embodiments, the forward sequencingstep comprises: a) contacting a plurality of sequencing polymerases to(i) a plurality of immobilized concatemer template molecules and (ii) aplurality of the soluble forward sequencing primers, wherein thecontacting is conducted under a condition suitable to form a pluralityof complexed polymerases each comprising a sequencing polymerase boundto a nucleic acid duplex wherein the nucleic acid duplex comprises aimmobilized concatemer template molecule hybridized to a soluble forwardsequencing primer; b) contacting the plurality of complexed sequencingpolymerases with a plurality of nucleotides under a condition suitablefor binding at least one nucleotide to a complexed sequencingpolymerase, wherein the plurality of nucleotides comprises at least onenucleotide analog labeled with a fluorophore and having a removablechain terminating moiety at the sugar 3′ position; c) incorporating atleast one nucleotide into the 3′ end of the hybridized forwardsequencing primers thereby generating a plurality of nascent extendedforward sequencing primers; and d) detecting the incorporated nucleotideand identifying the nucleo-base of the incorporated nucleotide.

In any of the foregoing or related embodiments, the reverse sequencingstep comprises: a) contacting a plurality of sequencing polymerases to(i) a plurality of the retained forward extension strands and (ii) aplurality of the soluble reverse sequencing primers, wherein thecontacting is conducted under a condition suitable to form a pluralityof complexed polymerases each comprising a sequencing polymerase boundto a nucleic acid duplex wherein the nucleic acid duplex comprises aretained forward extension strand hybridized to a soluble reversesequencing primer; b) contacting the plurality of complexed sequencingpolymerases with a plurality of nucleotides under a condition suitablefor binding at least one nucleotide to a complexed sequencingpolymerase, wherein the plurality of nucleotides comprises at least onenucleotide analog labeled with a fluorophore and having a removablechain terminating moiety at the sugar 3′ position; c) incorporating atleast one nucleotide into the 3′ end of the hybridized reversesequencing primers thereby generating a plurality of nascent extendedreverse sequencing primers; and d) detecting the incorporated nucleotideand identifying the nucleo-base of the incorporated nucleotide.

In some embodiments, the reverse sequencing of step (a) compriseshybridizing the plurality of soluble reverse sequencing primers to theplurality of the retained forward extension strands in the presence of ahigh efficiency hybridization buffer which comprises: (i) a first polaraprotic solvent which comprises acetonitrile at 25-50% by volume of thehybridization buffer; (ii) a second polar aprotic solvent whichcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) a pH buffering system which comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) acrowding agent which comprises polyethylene glycol (PEG) at 5-35% byvolume of the hybridization buffer.

In some embodiments, the reverse sequencing step comprises: a)contacting a plurality of sequencing polymerases to (i) a plurality ofthe immobilized partially displaced forward extension strands, (ii) aplurality of plurality of immobilized detached extended forwardsequencing primer strands, and (iii) a plurality of the soluble reversesequencing primers, wherein the contacting is conducted under acondition suitable to form a plurality of complexed polymerases eachcomprising a sequencing polymerase bound to a nucleic acid duplexwherein the nucleic acid duplex comprises a soluble reverse sequencingprimer hybridized to an immobilized partially displaced forwardextension strand or an immobilized detached extended forward sequencingprimer strand; b) contacting the plurality of complexed sequencingpolymerases with a plurality of nucleotides under a condition suitablefor binding at least one nucleotide to a complexed sequencingpolymerase, wherein the plurality of nucleotides comprises at least onenucleotide analog labeled with a fluorophore and having a removablechain terminating moiety at the sugar 3′ position; c) incorporating atleast one nucleotide into the 3′ end of the hybridized reversesequencing primers thereby generating a plurality of nascent extendedreverse sequencing primers; and d) detecting the incorporated nucleotideand identifying the nucleo-base of the incorporated nucleotide.

In some embodiments, the reverse sequencing of step a) compriseshybridizing the plurality of soluble reverse sequencing primers to theplurality of the retained forward extension strands in the presence of ahigh efficiency hybridization buffer which comprises: (i) a first polaraprotic solvent which comprises acetonitrile at 25-50% by volume of thehybridization buffer; (ii) a second polar aprotic solvent whichcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) a pH buffering system which comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) acrowding agent which comprises polyethylene glycol (PEG) at 5-35% byvolume of the hybridization buffer.

In any of the foregoing or related embodiments, the forward sequencingstep and the reverse sequencing step comprises: 1) conducting asequencing reaction at a position on the template molecule usingmultivalent molecules which bind but do not incorporate; 2) conducting asequencing reaction at the same position on the template molecule usingnucleotides with incorporation; and 3) repeating steps a) and b) at thenext position on the template molecule.

In any of the foregoing or related embodiments, the forward sequencingstep and the reverse sequencing step comprises: a) contacting aplurality of a first sequencing polymerase to (i) a plurality of nucleicacid template molecules and (ii) a plurality of soluble sequencingprimers, wherein the contacting is conducted under a condition suitableto form a plurality of first complexed polymerases each comprising afirst sequencing polymerase bound to a nucleic acid duplex wherein thenucleic acid duplex comprises the nucleic acid template moleculehybridized to the sequencing primer, wherein (1) the plurality ofnucleic acid template molecules comprise a plurality of the immobilizedconcatemer template molecules and the plurality of soluble primerscomprise a plurality of the soluble forward sequencing primers, orwherein (2) the plurality of nucleic acid template molecules comprise aplurality of the retained forward extension strands and the plurality ofsoluble sequencing primers comprise a plurality of the soluble reversesequencing primers; b) contacting the plurality of first complexedpolymerases with a plurality of detectably labeled multivalent moleculesto form a plurality of multivalent-complexed polymerases, under acondition suitable for binding complementary nucleotide units of themultivalent molecules to at least two of the plurality of firstcomplexed polymerases thereby forming a plurality ofmultivalent-complexed polymerases, and the condition inhibitsincorporation of the complementary nucleotide units into the sequencingprimers of the plurality of multivalent-complexed polymerases, whereinindividual multivalent molecules in the plurality of multivalentmolecules comprise a core attached to multiple nucleotide arms and eachnucleotide arm is attached to a nucleotide unit; c) detecting theplurality of multivalent-complexed polymerases; and d) identifying thenucleo-base of the complementary nucleotide units that are bound to theplurality of first complexed polymerases in the plurality ofmultivalent-complexed polymerases, thereby determining the sequence ofthe nucleic acid template.

In some embodiments, the reverse sequencing of step comprises:hybridizing the plurality of soluble reverse sequencing primers to theplurality of the retained forward extension strands in the presence of ahigh efficiency hybridization buffer which comprises: (i) a first polaraprotic solvent which comprises acetonitrile at 25-50% by volume of thehybridization buffer; (ii) a second polar aprotic solvent whichcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) a pH buffering system which comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) acrowding agent which comprises polyethylene glycol (PEG) at 5-35% byvolume of the hybridization buffer.

In some embodiments, the method further comprises: e) dissociating theplurality of multivalent-complexed polymerases and removing theplurality of first sequencing polymerases and their bound multivalentmolecules, and retaining the plurality of nucleic acid duplexes; f)contacting the plurality of the retained nucleic acid duplexes of step(e) with a plurality of second sequencing polymerases, wherein thecontacting is conducted under a condition suitable for binding theplurality of second sequencing polymerases to the plurality of theretained nucleic acid duplexes, thereby forming a plurality of secondcomplexed polymerases each comprising a second sequencing polymerasebound to a retained nucleic acid duplex; g) contacting the plurality ofsecond complexed polymerases with a plurality of nucleotides, whereinthe contacting is conducted under a condition suitable for bindingcomplementary nucleotides from the plurality of nucleotides to at leasttwo of the second complexed polymerases of step (f) thereby forming aplurality of nucleotide-complexed polymerases and the condition issuitable for promoting incorporation of the bound complementarynucleotides into the sequencing primers of the nucleotide-complexedpolymerases; h) detecting the complementary nucleotides which areincorporated into the sequencing primers of the nucleotide-complexedpolymerases; and d) identifying the nucleo-bases of the complementarynucleotides which are incorporated into the sequencing primers of thenucleotide-complexed polymerases.

In some embodiments, the method further comprises: forming at least oneavidity complex in step (b), the method comprising: a) binding a firstsequencing primer, a first sequencing polymerase, and a firstmultivalent molecule to a first portion of a nucleic acid templatemolecule thereby forming a first binding complex, wherein a firstnucleotide unit of the first multivalent molecule binds to the firstsequencing polymerase; and b) binding a second sequencing primer, asecond sequencing polymerase, and the first multivalent molecule to asecond portion of the same nucleic acid template molecule therebyforming a second binding complex, wherein a second nucleotide unit ofthe second multivalent molecule binds to the second sequencingpolymerase, wherein the first and second binding complexes which includethe same multivalent molecule forms an avidity complex.

In some embodiments, (i) the first sequencing primer comprises a solubleforward sequencing primer and the nucleic acid template moleculecomprises an immobilized concatemer template molecule, (ii) the secondsequencing primer comprises a soluble forward sequencing primer and thenucleic acid template molecule comprises the same immobilized concatemertemplate molecule, and (iii) the first and second sequencing primershave the same sequence.

In some embodiments, wherein (i) the first sequencing primer comprises asoluble reverse sequencing primer and the nucleic acid template moleculecomprises a retained forward extension strand, (ii) the secondsequencing primer comprises a soluble reverse sequencing primer and thenucleic acid template molecule comprises the same retained forwardextension strand, and (iii) the first and second sequencing primers havethe same sequence.

In some embodiments, the method further comprises: forming at least oneavidity complex in step (b), the method comprising: a) contacting aplurality of first sequencing polymerases and a plurality of secondsequencing primers with different portions of a nucleic acid templatemolecule to form at least first and second complexed polymerases on thesame nucleic acid template molecule; b) contacting a plurality ofmultivalent molecules to the at least first and second complexedpolymerases on the same nucleic acid template molecule, under conditionssuitable to bind a single multivalent molecule from the plurality to thefirst and second complexed polymerases, wherein at least a firstnucleotide unit of the single multivalent molecule is bound to the firstcomplexed polymerase which includes a first sequencing primer hybridizedto a first portion of the nucleic acid template molecule thereby forminga first binding complex, and wherein at least a second nucleotide unitof the single multivalent molecule is bound to the second complexedpolymerase which includes a second sequencing primer hybridized to asecond portion of the same nucleic acid template molecule therebyforming a second binding complex, wherein the contacting is conductedunder a condition suitable to inhibit polymerase-catalyzed incorporationof the bound first and second nucleotide units in the first and secondbinding complexes, and wherein the first and second binding complexeswhich are bound to the same multivalent molecule forms an aviditycomplex; c) detecting the first and second binding complexes on the samenucleic acid template molecule, and d) identifying the first nucleotideunit in the first binding complex thereby determining the sequence ofthe first portion of the nucleic acid template molecule, and identifyingthe second nucleotide unit in the second binding complex therebydetermining the sequence of the second portion of the same nucleic acidtemplate molecule.

In some embodiments, (i) the plurality of first sequencing primerscomprise a plurality of first soluble forward sequencing primers and thenucleic acid template molecule comprises an immobilized concatemertemplate molecule, (ii) the plurality of second sequencing primerscomprise a plurality of second soluble forward sequencing primers andthe nucleic acid template molecule comprises the same immobilizedconcatemer template molecule, and (iii) the plurality of first andsecond sequencing primers have the same sequence.

In some embodiments, (i) the plurality of first sequencing primerscomprises a plurality of first soluble reverse sequencing primer and thenucleic acid template molecule comprises a retained forward extensionstrand, (ii) the plurality of second sequencing primers comprise aplurality of second soluble reverse sequencing primers and the nucleicacid template molecule comprises the same retained forward extensionstrand, and (iii) the plurality of first and second sequencing primershave the same sequence.

In any of the foregoing or related embodiments, the forward sequencingstep and the reverse sequencing step comprises: a) contacting aplurality of a first sequencing polymerase to (i) a plurality of nucleicacid template molecules and (ii) a plurality of soluble sequencingprimers, wherein the contacting is conducted under a condition suitableto form a plurality of first complexed polymerases each comprising afirst sequencing polymerase bound to a nucleic acid duplex wherein thenucleic acid duplex comprises the nucleic acid template moleculehybridized to the soluble sequencing primer, wherein (1) the pluralityof nucleic acid template molecules comprise a plurality of theimmobilized concatemer template molecules and the plurality ofsequencing primers comprise a plurality of the soluble forwardsequencing primers, or wherein (2) the plurality of nucleic acidtemplate molecules comprise a plurality of immobilized partiallydisplaced forward extension strands and the plurality of sequencingprimers comprise a plurality of the soluble reverse sequencing primers,or wherein (3) the plurality of nucleic acid template molecules comprisea plurality of immobilized detached extended forward sequencing primerstrands and the plurality of sequencing primers comprise a plurality ofthe soluble reverse sequencing primers; b) contacting the plurality offirst complexed polymerases with a plurality of detectably labeledmultivalent molecules to form a plurality of multivalent-complexedpolymerases, under a condition suitable for binding complementarynucleotide units of the multivalent molecules to at least two of theplurality of first complexed polymerases, thereby forming a plurality ofmultivalent-complexed polymerases, and the condition inhibitsincorporation of the complementary nucleotide units into the sequencingprimers of the plurality of multivalent-complexed polymerases, whereinindividual multivalent molecules in the plurality of multivalentmolecules comprise a core attached to multiple nucleotide arms and eachnucleotide arm is attached to a nucleotide unit; c) detecting theplurality of multivalent-complexed polymerases; and d) identifying thenucleo-base of the complementary nucleotide units that are bound to theplurality of first complexed polymerases in the plurality ofmultivalent-complexed polymerases, thereby determining the sequence ofthe nucleic acid template.

In any of the foregoing or related embodiments, the reverse sequencingstep comprises: hybridizing the plurality of soluble reverse sequencingprimers to the plurality of immobilized partially displaced forwardextension strands or the plurality of immobilized detached extendedforward sequencing primer strands in the presence of a high efficiencyhybridization buffer which comprises: (i) a first polar aprotic solventwhich comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) a second polar aprotic solvent which comprises formamide at5-10% by volume of the hybridization buffer; (iii) a pH buffering systemwhich comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) a crowding agent which comprises polyethylene glycol(PEG) at 5-35% by volume of the hybridization buffer.

In some embodiments, the method further comprises: e) dissociating theplurality of multivalent-complexed polymerases and removing theplurality of first sequencing polymerases and their bound multivalentmolecules, and retaining the plurality of nucleic acid duplexes; f)contacting the plurality of the retained nucleic acid duplexes of step(e) with a plurality of second sequencing polymerases, wherein thecontacting is conducted under a condition suitable for binding theplurality of second sequencing polymerases to the plurality of theretained nucleic acid duplexes, thereby forming a plurality of secondcomplexed polymerases each comprising a second sequencing polymerasebound to a retained nucleic acid duplex; g) contacting the plurality ofsecond complexed polymerases with a plurality of nucleotides, whereinthe contacting is conducted under a condition suitable for bindingcomplementary nucleotides from the plurality of nucleotides to at leasttwo of the second complexed polymerases of step (f) thereby forming aplurality of nucleotide-complexed polymerases and the condition issuitable for promoting incorporation of the bound complementarynucleotides into the sequencing primers of the nucleotide-complexedpolymerases; h) detecting the complementary nucleotides which areincorporated into the sequencing primers of the nucleotide-complexedpolymerases; and i) identifying the nucleo-bases of the complementarynucleotides which are incorporated into the sequencing primers of thenucleotide-complexed polymerases.

In some embodiments, the method further comprises: forming at least oneavidity complex in step (b), the method comprising: a) binding a firstsequencing primer, a first sequencing polymerase, and a firstmultivalent molecule to a first portion of a nucleic acid templatemolecule thereby forming a first binding complex, wherein a firstnucleotide unit of the first multivalent molecule binds to the firstsequencing polymerase; and b) binding a second sequencing primer, asecond sequencing polymerase, and the first multivalent molecule to asecond portion of the same nucleic acid template molecule therebyforming a second binding complex, wherein a second nucleotide unit ofthe second multivalent molecule binds to the second sequencingpolymerase, wherein the first and second binding complexes which includethe same multivalent molecule forms an avidity complex.

In some embodiments, (i) the first sequencing primer comprises a solubleforward sequencing primer and the nucleic acid template moleculecomprises an immobilized concatemer template molecule, (ii) the secondsequencing primer comprises a soluble forward sequencing primer and thenucleic acid template molecule comprises the same immobilized concatemertemplate molecule, and (iii) the first and second sequencing primershave the same sequence. In some embodiments, (i) the first sequencingprimer comprises a soluble reverse sequencing primer and the nucleicacid template molecule comprises an immobilized partially displacedforward extension strand, (ii) the second sequencing primer comprises asoluble reverse sequencing primer and the nucleic acid template moleculecomprises the same immobilized partially displaced forward extensionstrand, and (iii) the first and second sequencing primers have the samesequence. In some embodiments, (i) the first sequencing primer comprisesa soluble reverse sequencing primer and the nucleic acid templatemolecule comprises an immobilized detached extended forward sequencingprimer strand, (ii) the second sequencing primer comprises a solublereverse sequencing primer and the nucleic acid template moleculecomprises the same immobilized detached extended forward sequencingprimer strand, and (iii) the first and second sequencing primers havethe same sequence.

In some embodiments, the method further comprises: forming at least oneavidity complex in step (b), the method comprising: a) contacting aplurality of first sequencing polymerases and a plurality of secondsequencing primers with different portions of a nucleic acid templatemolecule to form at least first and second complexed polymerases on thesame nucleic acid template molecule; b) contacting a plurality ofmultivalent molecules to the at least first and second complexedpolymerases on the same nucleic acid template molecule, under conditionssuitable to bind a single multivalent molecule from the plurality to thefirst and second complexed polymerases, wherein at least a firstnucleotide unit of the single multivalent molecule is bound to the firstcomplexed polymerase which includes a first sequencing primer hybridizedto a first portion of the nucleic acid template molecule thereby forminga first binding complex, and wherein at least a second nucleotide unitof the single multivalent molecule is bound to the second complexedpolymerase which includes a second sequencing primer hybridized to asecond portion of the same nucleic acid template molecule therebyforming a second binding complex, wherein the contacting is conductedunder a condition suitable to inhibit polymerase-catalyzed incorporationof the bound first and second nucleotide units in the first and secondbinding complexes, and wherein the first and second binding complexeswhich are bound to the same multivalent molecule forms an aviditycomplex; c) detecting the first and second binding complexes on the samenucleic acid template molecule, and d) identifying the first nucleotideunit in the first binding complex thereby determining the sequence ofthe first portion of the nucleic acid template molecule, and identifyingthe second nucleotide unit in the second binding complex therebydetermining the sequence of the second portion of the same nucleic acidtemplate molecule.

In some embodiments, (i) the plurality of first sequencing primerscomprise a plurality of first soluble forward sequencing primers and thenucleic acid template molecule comprises an immobilized concatemertemplate molecule, (ii) the plurality of second sequencing primerscomprise a plurality of second soluble forward sequencing primers andthe nucleic acid template molecule comprises the same immobilizedconcatemer template molecule, and (iii) the plurality of first andsecond sequencing primers have the same sequence. In some embodiments,(i) the plurality of first sequencing primers comprises a plurality offirst soluble reverse sequencing primer and the nucleic acid templatemolecule comprises an immobilized partially displaced forward extensionstrand, (ii) the plurality of second sequencing primers comprise aplurality of second soluble reverse sequencing primers and the nucleicacid template molecule comprises the same immobilized partiallydisplaced forward extension strand, and (iii) the plurality of first andsecond sequencing primers have the same sequence. In some embodiments,(i) the plurality of first sequencing primers comprises a plurality offirst soluble reverse sequencing primer and the nucleic acid templatemolecule comprises an immobilized detached extended forward sequencingprimer strands, (ii) the plurality of second sequencing primers comprisea plurality of second soluble reverse sequencing primers and the nucleicacid template molecule comprises the same immobilized detached extendedforward sequencing primer strands, and (iii) the plurality of first andsecond sequencing primers have the same sequence.

In any of the foregoing or related embodiments, individual nucleotidesin the plurality of nucleotides comprise an aromatic base, a five carbonsugar, and 1-10 phosphate groups, wherein the aromatic base of thenucleotide comprises adenine, guanine, cytosine, thymine or uracil. Insome embodiments, the plurality of nucleotides comprises one type ofnucleotide selected from a group consisting of dATP, dGTP, dCTP anddTTP. In some embodiments, the plurality of nucleotides comprises amixture of any combination of two or more types of nucleotides selectedfrom a group consisting of dATP, dGTP, dCTP and/or dTTP. In someembodiments, at least one of the nucleotides in the plurality ofnucleotides comprises a fluorescently-labeled nucleotide. In someembodiments, at least one of the plurality of nucleotides lacks afluorophore label.

In any of the foregoing or related embodiments, at least one of thenucleotides in the plurality of nucleotides comprises a chainterminating moiety attached to 3′-OH sugar position via cleavablemoiety, and wherein the chain terminating moiety comprises an alkylgroup, alkenyl group, alkynyl group, allyl group, aryl group, benzylgroup, azide group, amine group, amide group, keto group, isocyanategroup, phosphate group, thio group, disulfide group, carbonate group,urea group, or silyl group.

In some embodiments, the chain terminating moieties alkyl, alkenyl,alkynyl and allyl are cleavable/removable withtetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, orwith 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (i) the chainterminating moieties aryl and benzyl are cleavable/removable with H2Pd/C; (ii) the chain terminating moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable/removable with athiol reagent which comprises beta-mercaptoethanol or dithiothritol(DTT); (iii) the chain terminating moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable/removable with aphosphine reagent which comprises Tris(2-carboxyethyl)phosphine (TCEP),bis-sulfo triphenyl phosphine (BS-TPP), or Tri(hydroxyproyl)phosphine(THPP); (iv) the chain terminating moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable/removable with4-dimethylaminopyridine (4-DMAP); (v) the chain terminating moietycarbonate is cleavable/removable with potassium carbonate (K2CO3) inMeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH);and (vi) the chain terminating moieties urea and silyl are cleavablewith tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride,or with triethylamine trihydrofluoride.

In some embodiments, at least one of the nucleotides in the plurality ofnucleotides comprises a chain terminating moiety attached to 3′-OH sugarposition via cleavable moiety, and wherein the chain terminating moietycomprises a 3′ O-azido or a 3′ O-azidomethyl group. In some embodiments,(i) the chain terminating moieties 3′ O-azido and 3′ O-azidomethyl groupare cleavable/removable with a phosphine compound which comprise aderivatized tri-alkyl phosphine moiety, derivatized tri-aryl phosphinemoiety, Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenylphosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP); and (ii) thechain terminating moieties 3′ O-azido and 3′ O-azidomethyl group arecleavable/removable with 4-dimethylaminopyridine (4-DMAP).

In any of the foregoing or related embodiments, individual multivalentmolecules in the plurality of multivalent molecules comprises (a) acore; and (b) a plurality of nucleotide arms which comprise (i) a coreattachment moiety, (ii) a spacer comprising a PEG moiety, (iii) alinker, and (iv) a nucleotide unit, wherein the core is attached to theplurality of nucleotide arms via their core attachment moiety, whereinthe spacer is attached to the linker, and wherein the linker is attachedto the nucleotide unit.

In some embodiments, the core comprises an avidin-type moiety and thecore attachment moiety comprises biotin. In some embodiments, the linkercomprises an aliphatic chain having 2-6 subunits or an oligo ethyleneglycol chain having 2-6 subunits. In some embodiments, the linkerfurther comprises an aromatic moiety. In some embodiments, thenucleotide unit comprises an aromatic base, a five carbon sugar and 1-10phosphate groups. In some embodiments, the linker is attached to thenucleotide unit through the base.

In some embodiments, the plurality of nucleotide arms attached to thecore have the same type of a nucleotide unit, and wherein the types ofnucleotide unit is selected from a group consisting of dATP, dGTP, dCTP,dTTP and dUTP. In some embodiments, the plurality of multivalentmolecules comprise one type of a multivalent molecule wherein eachmultivalent molecule in the plurality has the same type of nucleotideunit selected from a group consisting of dATP, dGTP, dCTP, dTTP anddUTP. In some embodiments, the plurality of multivalent moleculescomprise a mixture of any combination of two or more types ofmultivalent molecules each type having nucleotide units selected from agroup consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

In some embodiments, the plurality of multivalent molecules arefluorescently-labeled multivalent molecules. In some embodiments, (i)the core of individual fluorescently-labeled multivalent molecules isattached to a fluorophore which corresponds to the nucleotide units thatare attached to the nucleotide arms; (ii) at least one of the nucleotidearms comprises a linker that is attached to a fluorophore whichcorresponds to the nucleotide units that are attached to the nucleotidearms; and/or (iii) at least one of the nucleotide arms comprises anucleotide unit that is attached to a fluorophore which corresponds tothe nucleotide units that are attached to the nucleotide arms.

In some embodiments, the plurality of multivalent molecules lack afluorophore.

In some embodiments, at least one of the multivalent molecules in theplurality of multivalent molecules comprises nucleotide units having achain terminating moiety attached to the 3′-OH sugar position via acleavable moiety, and wherein the chain terminating moiety comprises analkyl group, alkenyl group, alkynyl group, allyl group, aryl group,benzyl group, azide group, amine group, amide group, keto group,isocyanate group, phosphate group, thio group, disulfide group,carbonate group, urea group, or silyl group.

In some embodiments, (i) the chain terminating moieties alkyl, alkenyl,alkynyl and allyl are cleavable/removable withtetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, orwith 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (ii) the chainterminating moieties aryl and benzyl are cleavable/removable with H2Pd/C; (iii) the chain terminating moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable/removable with athiol reagent which comprises beta-mercaptoethanol or dithiothritol(DTT); (iv) the chain terminating moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable/removable with aphosphine reagent which comprises Tris(2-carboxyethyl)phosphine (TCEP),bis-sulfo triphenyl phosphine (BS-TPP), or Tri(hydroxyproyl)phosphine(THPP); (v) the chain terminating moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable/removable with4-dimethylaminopyridine (4-DMAP); (vi) the chain terminating moietycarbonate is cleavable/removable with potassium carbonate (K2CO3) inMeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH);and (vii) the chain terminating moieties urea and silyl are cleavablewith tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride,or with triethylamine trihydrofluoride.

In some embodiments, at least one of the multivalent molecules in theplurality of multivalent molecules comprises nucleotide units having achain terminating moiety attached to the 3′-OH sugar position via acleavable moiety, and wherein the chain terminating moiety comprises a3′ O-azido or 3′ O-azidomethyl group.

In some embodiments, (i) the chain terminating moieties 3′ O-azido and3′ O-azidomethyl group are cleavable/removable with a phosphine compoundwhich comprise a derivatized tri-alkyl phosphine moiety, derivatizedtri-aryl phosphine moiety, Tris(2-carboxyethyl)phosphine (TCEP),bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine(THPP); and (ii) the chain terminating moieties 3′ O-azido and 3′O-azidomethyl are cleavable/removable with 4-dimethylaminopyridine(4-DMAP).

In some embodiments, the plurality of first sequencing polymerases ofstep (a) comprise a recombinant wild type DNA polymerase. In someembodiments, the plurality of first sequencing polymerases of step (a)comprise mutant DNA polymerase.

In some embodiments, the plurality of sequencing polymerases in step (a)comprises a recombinant wild type DNA polymerase, and the plurality ofnucleotides in step (b) comprises fluorescently-labeled nucleotideshaving a removable chain terminating moiety at the 3′ sugar position. Insome embodiments, the plurality of sequencing polymerases in step (a)comprises a mutant DNA polymerase, and the plurality of nucleotides instep (b) comprises fluorescently-labeled nucleotides having a removablechain terminating moiety at the 3′ sugar position.

In some embodiments, the plurality of second sequencing polymerases ofstep (f) comprise recombinant wild type DNA polymerase, and theplurality of nucleotides in step (b) comprises fluorescently-labelednucleotides having a removable chain terminating moiety at the 3′ sugarposition. In some embodiments, the plurality of second sequencingpolymerases of step (f) comprise mutant DNA polymerase, and theplurality of nucleotides in step (b) comprises fluorescently-labelednucleotides having a removable chain terminating moiety at the 3′ sugarposition.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands with a pluralityof forward extension strands that are hybridized to the retainedimmobilized single stranded nucleic acid concatemer template moleculesby conducting a primer extension reaction comprises: (i) contacting atleast one extended forward sequencing primer strand with a plurality ofstrand displacing polymerases and a plurality of nucleotides and in theabsence of soluble amplification primers, under a condition suitable toconduct a strand displacing primer extension reaction using the at leastone extended forward sequencing primers strand to initiate the primerextension reaction thereby generating a forward extension strand that iscovalently joined to the extended forward sequencing primers strand,wherein the forward extension strand is hybridized to the immobilizedconcatemer template molecule.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands with a pluralityof forward extension strands that are hybridized to the retainedimmobilized single stranded nucleic acid concatemer template moleculesby conducting a primer extension reaction comprises removing theplurality of extended forward sequencing primer strands by: (i)contacting the plurality of extended forward sequencing primer strandswith a 5′ to 3′ double-stranded DNA exonuclease; (ii) contacting theplurality of extended forward sequencing primer strands with adenaturation reagent comprising any combination of formamide,acetonitrile, guanidinium chloride and/or a pH buffering agent; or (iii)contacting the plurality of extended forward sequencing primer strandswith 100% formamide.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands with a pluralityof forward extension strands that are hybridized to the retainedimmobilized single stranded nucleic acid concatemer template moleculesby conducting a primer extension reaction comprises: (i) removing theplurality of extended forward sequencing primer strands while retainingthe immobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a secondplurality of soluble forward sequencing primers, a plurality ofnucleotides and a plurality of primer extension polymerases, under acondition suitable to hybridize the plurality of soluble forwardsequencing primers to the plurality of retained immobilized concatemertemplate molecules and suitable for conducting polymerase-catalyzedprimer extension reactions thereby generating a plurality of forwardextension strands, wherein the plurality of nucleotides comprise dATP,dGTP, dCTP and dTTP but lacks dUTP, wherein in the plurality of primerextension polymerases are tolerant of uridine-containing templatestrands, and wherein the soluble sequencing primers hybridize with theforward sequencing primer binding sequence in the retained immobilizedconcatemer molecules.

In some embodiments, the contacting comprises: contacting the pluralityof retained immobilized concatemer molecules with the plurality ofsoluble forward sequencing primers in the presence of a high efficiencyhybridization buffer which comprises: (i) a first polar aprotic solventwhich comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) a second polar aprotic solvent which comprises formamide at5-10% by volume of the hybridization buffer; (iii) a pH buffering systemwhich comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) a crowding agent which comprises polyethylene glycol(PEG) at 5-35% by volume of the hybridization buffer.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands with a pluralityof forward extension strands that are hybridized to the retainedimmobilized single stranded nucleic acid concatemer template moleculesby conducting a primer extension reaction comprises: (i) removing theplurality of extended forward sequencing primer strand while retainingthe immobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides and aplurality of primer extension polymerases, under a condition suitable tohybridize the plurality of soluble amplification primers to theplurality of retained immobilized concatemer template molecules andsuitable for conducting polymerase-catalyzed primer extension reactionsthereby generating a plurality of forward extension strands, wherein thesoluble amplification primers hybridize with the soluble amplificationprimer binding sequence in the retained immobilized concatemermolecules, wherein the plurality of nucleotides comprise dATP, dGTP,dCTP and dTTP but lacks dUTP, wherein in the plurality of primerextension polymerases are tolerant of uridine-containing templatestrands, and wherein the soluble sequencing primers hybridize with theforward sequencing primer binding sequence in the retained immobilizedconcatemer molecules.

In some embodiments, the contacting comprises: contacting the pluralityof retained immobilized concatemer molecules with the plurality ofsoluble amplification primers in the presence of a high efficiencyhybridization buffer which comprises: (i) a first polar aprotic solventwhich comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) a second polar aprotic solvent which comprises formamide at5-10% by volume of the hybridization buffer; (iii) a pH buffering systemwhich comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) a crowding agent which comprises polyethylene glycol(PEG) at 5-35% by volume of the hybridization buffer.

In some embodiments, the method further comprises: contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble compaction oligonucleotides.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands comprises: (i)contacting at least one extended forward sequencing primer strand with aplurality of strand displacing polymerases and a plurality ofnucleotides and in the absence of soluble amplification primers, under acondition suitable to conduct a strand displacing primer extensionreaction using the at least one extended forward sequencing primerstrand to initiate the primer extension reaction thereby generating aplurality of forward extension strands, a plurality of partiallydisplaced extended forward sequencing strands and a plurality ofdetached extended forward sequencing primer strands.

In any of the foregoing or related embodiments, replacing the pluralityof extended forward sequencing primer strands comprises: comprisesremoving the plurality of extended forward sequencing primer strands by:(i) contacting the plurality of extended forward sequencing primerstrands with a 5′ to 3′ double-stranded DNA exonuclease; (ii) contactingthe plurality of extended forward sequencing primer strands with adenaturation reagent comprising any combination of formamide,acetonitrile, guanidinium chloride and/or a pH buffering agent; or (iii)contacting the plurality of extended forward sequencing primer strandswith 100% formamide.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands comprises: (i)removing the plurality of extended forward sequencing primer strandswhile retaining the immobilized concatemer template molecules; and (ii)contacting the plurality of retained immobilized concatemer moleculeswith a second plurality of soluble forward sequencing primers, aplurality of nucleotides and a plurality of strand displacingpolymerases, under a condition suitable to hybridize the plurality ofsoluble forward sequencing primers to the plurality of retainedimmobilized concatemer template molecules and suitable for conductingpolymerase-catalyzed strand displacing reactions thereby generating aplurality of forward extension strands and a plurality of partiallydisplaced extended forward sequencing strands that are hybridized to theimmobilized concatemer template molecules to form a plurality ofimmobilized amplicons, and the primer extension reaction generates aplurality of detached extended forward sequencing primer strands (e.g.,that are not hybridized to the immobilized concatemer templatemolecules), wherein the plurality of nucleotides comprise dATP, dGTP,dCTP and dTTP but lacks dUTP, and wherein the soluble forward sequencingprimers hybridize with the forward sequencing primer binding sequence inthe retained immobilized concatemer molecules.

In some embodiments, the contacting comprises: contacting the pluralityof retained immobilized concatemer molecules with the plurality ofsoluble forward sequencing primers in the presence of a high efficiencyhybridization buffer which comprises: (i) a first polar aprotic solventwhich comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) a second polar aprotic solvent which comprises formamide at5-10% by volume of the hybridization buffer; (iii) a pH buffering systemwhich comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) a crowding agent which comprises polyethylene glycol(PEG) at 5-35% by volume of the hybridization buffer.

In any of the foregoing or related embodiments, the replacing theplurality of extended forward sequencing primer strands comprises: (i)removing the plurality of extended forward sequencing primer strandwhile retaining the immobilized concatemer template molecules; and (ii)contacting the plurality of retained immobilized concatemer moleculeswith a plurality of soluble amplification primers, a plurality ofnucleotides and a plurality of strand displacing polymerases, under acondition suitable to hybridize the plurality of soluble amplificationprimers to the plurality of retained immobilized concatemer templatemolecules and suitable for conducting polymerase-catalyzed stranddisplacing reactions thereby generating a plurality of forward extensionstrands and a plurality of partially displaced extended forwardsequencing strands that are hybridized to the immobilized concatemertemplate molecules to form a plurality of immobilized amplicons, and theprimer extension reaction generates a plurality of detached extendedforward sequencing primer strands (e.g., that are not hybridized to theimmobilized concatemer template molecules), wherein the plurality ofnucleotides comprise dATP, dGTP, dCTP and dTTP but lacks dUTP, whereinthe soluble amplification primers hybridize with the solubleamplification primer binding sequence in the retained immobilizedconcatemer molecules.

In some embodiments, the contacting comprises: contacting the pluralityof retained immobilized concatemer molecules with the plurality ofsoluble amplification primers in the presence of a high efficiencyhybridization buffer which comprises: (i) a first polar aprotic solventwhich comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) a second polar aprotic solvent which comprises formamide at5-10% by volume of the hybridization buffer; (iii) a pH buffering systemwhich comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of5-6.5; and (iv) a crowding agent which comprises polyethylene glycol(PEG) at 5-35% by volume of the hybridization buffer.

In any of the foregoing or related embodiments, the at least one of theretained immobilized concatemer template molecules includes one or morenucleotides having a scissile moiety, and wherein the scissile moietycomprises uridine or 8-oxo-7,8-dihydroguanine, or deoxyinosine. In anyof the foregoing or related embodiments, the retained immobilizedconcatemer template molecule comprises one or more uridines, and whereinthe generating the abasic sites at the uridines comprises contacting theretained immobilized concatemer template molecule with uracil DNAglycosylase (UDG). In any of the foregoing or related embodiments, theretained immobilized concatemer template molecule comprises one or more8oxoG, and wherein the generating the abasic sites at the 8oxoGcomprises contacting the retained immobilized concatemer templatemolecule with an Fpg enzyme (formamidopyrimidine DNA glycosylase). Inany of the foregoing or related embodiments, the retained immobilizedconcatemer template molecule comprises one or more deoxyinosine, andwherein the generating the abasic sites at the deoxyinosine comprisescontacting the retained immobilized concatemer template molecule with anAlkA glycosylase enzyme.

In any of the foregoing or related embodiments, the method furthercomprises generating a gap at the abasic sites to generate at least onegap-containing concatemer template molecule, which comprises: contactingthe retained immobilized template molecules containing one or moreabasic sites with an endonuclease IV, AP lyase (e.g., DNA-apurinic lyaseor DNA-apyrimidinic lyase), FPG glycosylase/AP lyase and/or endo VIIIglycosylase/AP lyase.

In any of the foregoing or related embodiments, the immobilizedconcatemer template molecules comprise 0.1-30% uridine, and wherein theplurality of wild type sequencing polymerases yield an error rate ofincorporating dUTP of at least 0.1× compared to an error rate ofincorporating dTTP. In any of the foregoing or related embodiments, theimmobilized concatemer template molecules comprise 0.1-30% uridine, andwherein the plurality of mutant sequencing polymerases yield an errorrate of incorporating dUTP of at least 0.1× compared to an error rate ofincorporating dTTP. In any of the foregoing or related embodiments, theimmobilized concatemer template molecules comprise 0.1-30% uridine, andwherein the plurality of wild type sequencing polymerases yield an errorrate of incorporating dUTP of at least 0.1× compared to an error rate ofincorporating dTTP. In any of the foregoing or related embodiments, theimmobilized concatemer template molecules comprise 0.1-30% uridine, andwherein the plurality of mutant sequencing polymerases yield an errorrate of incorporating dUTP of at least 0.1× compared to an error rate ofincorporating dTTP.

In any of the foregoing or related embodiments, the ratio of a firstbase fluorescent signal of R2 (e.g., reverse sequencing) to a first basefluorescent signal of R1 (e.g., forward sequencing) is at least 0.7 forsequencing using 1, 2, 3 or 4 dyes colors.

In any of the foregoing or related embodiments, the rolling circleamplification step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate immobilized concatemer template moleculeshaving a more compact size and/or shape compared to a rolling circleamplification reaction in the absence of compaction oligonucleotidesand/or hexamine.

In any of the foregoing or related embodiments, the primer extensionreaction of step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of forward extension strandshaving a more compact size and/or shape compared to a primer extensionreaction in the absence of compaction oligonucleotides and/or hexamine.

In any of the foregoing or related embodiments, the rolling circleamplification step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate concatemer molecules having a more compactsize and/or shape compared to a rolling circle amplification reaction inthe absence of compaction oligonucleotides and/or hexamine.

In any of the foregoing or related embodiments, the primer extensionreaction step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of forward extension strandshaving a more compact size and/or shape compared to a primer extensionreaction in the absence of compaction oligonucleotides and/or hexamine.

In any of the foregoing or related embodiments, the rolling circleamplification step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate immobilized concatemer template moleculeshaving a more compact size and/or shape compared to a rolling circleamplification reaction in the absence of compaction oligonucleotidesand/or hexamine.

In any of the foregoing or related embodiments, the primer extensionreaction step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of forward extension strandshaving a more compact size and/or shape compared to a primer extensionreaction in the absence of compaction oligonucleotides and/or hexamine.

In any of the foregoing or related embodiments, the primer extensionreaction step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of primer extension productshaving a more compact size and/or shape compared to a primer extensionreaction in the absence of compaction oligonucleotides and/or hexamine,wherein the plurality of primer extension products include a pluralityof forward extension strands, a plurality of partially displacedextended forward sequencing strands and a plurality of detached extendedforward sequencing primer strands.

In any of the foregoing or related embodiments, the rolling circleamplification step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate immobilized concatemer template moleculeshaving a more compact size and/or shape compared to a rolling circleamplification reaction in the absence of compaction oligonucleotidesand/or hexamine.

In any of the foregoing or related embodiments, the primer extensionreaction step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of primer extension productshaving a more compact size and/or shape compared to a primer extensionreaction in the absence of compaction oligonucleotides and/or hexamine,wherein the plurality of primer extension products include a pluralityof forward extension strands, a plurality of partially displacedextended forward sequencing strands and a plurality of detached extendedforward sequencing primer strands.

In any of the foregoing or related embodiments, the rolling circleamplification step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of concatemer molecules having amore compact size and/or shape compared to a rolling circleamplification reaction in the absence of compaction oligonucleotidesand/or hexamine.

In any of the foregoing or related embodiments, the primer extensionreaction step comprises a plurality of compaction oligonucleotidesand/or hexamine to generate a plurality of primer extension productshaving a more compact size and/or shape compared to a primer extensionreaction in the absence of compaction oligonucleotides and/or hexamine,wherein the plurality of primer extension products include a pluralityof forward extension strands, a plurality of partially displacedextended forward sequencing strands and a plurality of detached extendedforward sequencing primer strands.

In any of the foregoing or related embodiments, the plurality ofimmobilized concatemer template molecules or the plurality ofimmobilized concatemer molecules have FWHM (full width half maximum) ofno more than about 5 μm. In any of the foregoing or related embodiments,the plurality of forward extension strand have FWHM (full width halfmaximum) of no more than about 5 μm. In any of the foregoing or relatedembodiments, the plurality of primer extension products have FWHM (fullwidth half maximum) of no more than about 5 μm.

DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic showing an exemplary single stranded nucleic acidconcatemer template molecule immobilized to an immobilized first surfaceprimer. The immobilized concatemer template molecule comprises at leastone nucleotide having a scissile moiety that can be cleaved to generatean abasic site in the immobilized concatemer template molecule. In someembodiments, the immobilized concatemer template molecule can begenerated by conducting an on-support rolling circle amplificationreaction. The arrangement of the various primer binding sequences is forillustration purposes. The skilled artisan will appreciate that manyother arrangements are possible. FIGS. 2-12 show the workflow ofpairwise sequencing the immobilized concatemer template moleculedepicted in FIG. 1 .

FIG. 2 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.1 . The forward sequencing reaction can be conducted with a plurality ofsoluble forward sequencing primers and generates a plurality of extendedforward sequencing primer strands. The immobilized concatemer templatemolecule can have two or more extended forward sequencing primer strandshybridized thereon.

FIG. 3 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a strand displacing polymerase in the absence ofa soluble primer thereby generating a forward extension strand.

FIG. 4 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble forward sequencing primer therebygenerating a forward extension strand.

FIG. 5 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble amplification primer therebygenerating a forward extension strand.

FIG. 6 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 3 or 4 .

FIG. 7 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 6 .

FIG. 8 is a schematic showing an exemplary is a schematic showing anexemplary method for generating abasic sites in the immobilized singlestranded concatemer template molecules at the nucleotides having thescissile moiety and generating gaps at the abasic sites to generate aplurality of gap-containing concatemer template molecules whileretaining the plurality of forward extension strands and retaining theplurality of immobilized first surface primers. The forward extensionstrand can be generated by the method depicted in FIG. 5 .

FIG. 9 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 8 .

FIG. 10 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 7 . Thereverse sequencing reaction can be conducted with a plurality of solublereverse sequencing primers. The retained forward extension strand canhave two or more extended reverse sequencing primer strands hybridizedthereon. The extended reverse sequencing primer strands are nothybridized to the first surface primer, or covalently joined to thefirst surface primer. Therefore, the extended reverse sequencing primerstrands are not immobilized to the support. For the sake of simplicity,FIGS. 1-10 show an exemplary immobilized concatemer molecule with onecopy of the sequence of interest and various universal primer bindingsites. The skilled artisan will appreciate that the immobilizedconcatemer molecule can include two or more tandem copies containing thesequence of interest and various universal primer binding sites.

FIG. 11 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 9 . Theretained forward extension strand can have two or more extended reversesequencing primer strands hybridized thereon. The extended reversesequencing primer strands are not hybridized to the first surfaceprimer, or covalently joined to the first surface primer. Therefore, theextended reverse sequencing primer strands are not immobilized to thesupport. For the sake of simplicity, FIGS. 1-11 show an exemplaryimmobilized concatemer molecule with one copy of the sequence ofinterest and various universal primer binding sites. The skilled artisanwill appreciate that the immobilized concatemer molecule can include twoor more tandem copies containing the sequence of interest and variousuniversal primer binding sites.

FIG. 12 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 1 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 13 is a schematic showing an exemplary single stranded nucleic acidconcatemer template molecule immobilized to an immobilized first surfaceprimer. The immobilized concatemer template molecule comprises at leastone nucleotide having a scissile moiety that can be cleaved to generatean abasic site in the immobilized concatemer template molecule. In someembodiments, the immobilized concatemer template molecule can begenerated by conducting an in-solution rolling circle amplificationreaction and distributing the rolling circle amplification reaction ontothe support. The arrangement of the various primer binding sequences isfor illustration purposes. The skilled artisan will appreciate that manyother arrangements are possible. FIGS. 14-25 show the workflow ofpairwise sequencing the immobilized concatemer template moleculedepicted in FIG. 13 .

FIG. 14 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.13 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 15 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a strand displacing polymerase in the absence ofa soluble primer.

FIG. 16 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble forward sequencing primer.

FIG. 17 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble amplification primer.

FIG. 18 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 15 or 16 .

FIG. 19 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 18 .

FIG. 20 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 17 .

FIG. 21 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 20 .

FIG. 22 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 19 .The reverse sequencing reaction can be conducted with a plurality ofsoluble reverse sequencing primers. The retained forward extensionstrand depicted in FIG. 22 is a concatemer molecule that can include twoor more tandem copies of the sequence of interest and various primerbinding sites. Such a concatemer molecule can have two or more extendedreverse sequencing primer strands hybridized thereon. The extendedreverse sequencing primer strands are not hybridized to the firstsurface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support. For the sake of simplicity, FIGS. 13-23 showan exemplary immobilized concatemer molecule with one copy of thesequence of interest and various universal primer binding sites. Theskilled artisan will appreciate that the immobilized concatemer moleculecan include two or more tandem copies containing the sequence ofinterest and various universal primer binding sites.

FIG. 23 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 21 .The retained forward extension strand can have two or more extendedreverse sequencing primer strands hybridized thereon. The retainedforward extension strand depicted in FIG. 23 is a concatemer moleculethat includes two or more tandem copies of the sequence of interest andvarious primer binding sites. Such a concatemer molecule can have two ormore extended reverse sequencing primer strands hybridized thereon. Theextended reverse sequencing primer strands are not hybridized to thefirst surface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support. For the sake of simplicity, FIGS. 13-23 showan exemplary immobilized concatemer molecule with two tandem copiescontaining the sequence of interest and various universal primer bindingsites. The skilled artisan will appreciate that the immobilizedconcatemer molecule can include three or more tandem copies containingthe sequence of interest and various universal primer binding sites.

FIG. 24 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 13 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 25 is a schematic showing an exemplary support having a firstsurface primer immobilized thereon, which in some embodiments, can beused to conduct an on-support pairwise sequencing workflow.

FIG. 26 is a schematic showing an exemplary on-support rolling circleamplification reaction using a nucleic acid circular library molecule,the immobilized first surface primer shown in FIG. 25 , and a mixture ofnucleotides including nucleotides having a scissile moiety that can becleaved to generate an abasic site. The rolling circle amplificationreaction generates an immobilized single stranded nucleic acidconcatemer template molecule having at least one nucleotide with ascissile moiety which can be cleaved to generate an abasic site in theimmobilized concatemer template molecule. The arrangement of the variousprimer binding sequences in the nucleic acid circular library moleculeis for illustration purposes. The skilled artisan will appreciate thatmany other arrangements are possible. FIGS. 26-37 show the workflow ofpairwise sequencing the immobilized concatemer template moleculedepicted in FIG. 26 .

FIG. 27 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.26 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers and generates a plurality ofextended forward sequencing primer strands. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 28 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a strand displacing polymerase in the absence ofa soluble primer thereby generating a forward extension strand.

FIG. 29 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble forward sequencing primer therebygenerating a forward extension strand.

FIG. 30 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble amplification primer therebygenerating a forward extension strand.

FIG. 31 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 28 or 29 .

FIG. 32 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 31 .

FIG. 33 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 30 .

FIG. 34 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 33 .

FIG. 35 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 32 .The reverse sequencing reaction can be conducted with a plurality ofsoluble reverse sequencing primers. The retained forward extensionstrand can have two or more extended reverse sequencing primer strandshybridized thereon. The extended reverse sequencing primer strands arenot hybridized to the first surface primer, or covalently joined to thefirst surface primer. Therefore, the extended reverse sequencing primerstrands are not immobilized to the support. For the sake of simplicity,FIGS. 26-36 show an exemplary immobilized concatemer molecule with onecopy of the sequence of interest and various universal primer bindingsites. The skilled artisan will appreciate that the immobilizedconcatemer molecule can include two or more tandem copies containing thesequence of interest and various universal primer binding sites.

FIG. 36 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 34 .The retained forward extension strand can have two or more extendedreverse sequencing primer strands hybridized thereon. The extendedreverse sequencing primer strands are not hybridized to the firstsurface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support. For the sake of simplicity, FIGS. 26-36 showan exemplary immobilized concatemer molecule with one copy of thesequence of interest and various universal primer binding sites. Theskilled artisan will appreciate that the immobilized concatemer moleculecan include two or more tandem copies containing the sequence ofinterest and various universal primer binding sites.

FIG. 37 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 26 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 38 is a schematic showing an exemplary in-solution rolling circleamplification reaction using a nucleic acid circular library molecule, asoluble first amplification primer, and a mixture of nucleotidesincluding nucleotides having a scissile moiety that can be cleaved togenerate an abasic site. The rolling circle amplification reactiongenerates in solution single stranded nucleic acid concatemer moleculeshaving at least one nucleotide with a scissile moiety which can becleaved to generate an abasic site in the concatemer molecule. Thearrangement of the various primer binding sequences in the nucleic acidcircular library molecule is for illustration purposes. The skilledartisan will appreciate that many other arrangements are possible. FIGS.38-52 show the workflow of pairwise sequencing the concatemer moleculedepicted in FIG. 38 .

FIG. 39 is a schematic showing an exemplary method comprisingdistributing the rolling circle amplification reaction depicted in FIG.38 onto a support having a first surface primer immobilized thereon. Theconcatemer molecule can hybridize to the immobilized first surfaceprimer.

FIG. 40 is a schematic showing an exemplary method which depicts therolling circle amplification reaction continuing on the support therebygenerating an immobilized concatemer template molecule which includes atleast one nucleotide with a scissile moiety which can be cleaved togenerate an abasic site in the immobilized concatemer template molecule.

FIG. 41 is a schematic showing an exemplary immobilized concatemertemplate molecule generated by the method depicted in FIG. 40 .

FIG. 42 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.41 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 43 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a strand displacing polymerase in the absence ofa soluble primer.

FIG. 44 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble forward sequencing primer.

FIG. 45 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble amplification primer.

FIG. 46 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 43 or 44 .

FIG. 47 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 46 .

FIG. 48 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 45 .

FIG. 49 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 48 .

FIG. 50 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 47 .The reverse sequencing reaction can be conducted with a plurality ofsoluble reverse sequencing primers. The retained forward extensionstrand depicted in FIG. 50 is a concatemer molecule that can include twoor more tandem copies of the sequence of interest and various primerbinding sites. Such a concatemer molecule can have two or more extendedreverse sequencing primer strands hybridized thereon. The extendedreverse sequencing primer strands are not hybridized to the firstsurface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support. For the sake of simplicity, FIGS. 41-50 showan exemplary immobilized concatemer molecule with one copy of thesequence of interest and various universal primer binding sites. Theskilled artisan will appreciate that the immobilized concatemer moleculecan include two or more tandem copies containing the sequence ofinterest and various universal primer binding sites.

FIG. 51 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 49 .The retained forward extension strand can have two or more extendedreverse sequencing primer strands hybridized thereon. The retainedforward extension strand depicted in FIG. 51 is a concatemer moleculethat includes two or more tandem copies of the sequence of interest andvarious primer binding sites. Such a concatemer molecule can have two ormore extended reverse sequencing primer strands hybridized thereon. Theextended reverse sequencing primer strands are not hybridized to thefirst surface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support. For the sake of simplicity, FIGS. 41-51 showan exemplary immobilized concatemer molecule with two tandem copiescontaining the sequence of interest and various universal primer bindingsites. The skilled artisan will appreciate that the immobilizedconcatemer molecule can include three or more tandem copies containingthe sequence of interest and various universal primer binding sites.

FIG. 52 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 41 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 53 is schematic showing a linear single stranded library molecule(left top schematic) hybridizing with a double stranded splint molecule(left bottom schematic) to generate a circular library molecule with twogaps (right schematic). The splint molecule comprises a first splintstrand (long strand) hybridized to a second splint strand (shortstrand). The first splint strand comprises a left sequence thathybridizes with a sequence on one end of the linear single strandedlibrary molecule, and a right sequence that hybridizes with a sequenceon the other end of the linear single stranded library molecule. Theinterior portion of the first splint strand hybridizes to the secondsplint strand.

FIG. 54 is a schematic showing the circular library molecule (leftschematic) which is shown in FIG. 53 undergoing a ligation reaction togenerate a single stranded covalently closed circular molecule which ishybridized to the first splint strand (center schematic). The singlestranded covalently closed circular molecule is subjected to a rollingcircle amplification reaction using the 3′ end of the first splintstrand to initiate the RCA reaction (right schematic).

FIG. 55 is a schematic showing an exemplary support having a firstsurface primer immobilized thereon, which in some embodiments, can beused to conduct an on-support ligation reaction for a pairwisesequencing workflow. FIGS. 55-72 show the workflow of on-supportligation and pairwise sequencing.

FIG. 56 is a schematic showing an exemplary single stranded linearlibrary molecule comprising a sequence of interest and various universaladaptor sequences for primer binding sites. The arrangement of thevarious universal adaptor sequences in this schematic is forillustration purposes. The skilled artisan will appreciate that manyother arrangements, and combinations of universal adaptor sequences, arepossible.

FIG. 57 is a schematic showing an exemplary single stranded linearlibrary molecule hybridized to an immobilized first surface primer toform a circularized library molecule having an asymmetrically positionedgap or nick.

FIG. 58 (left) is a schematic showing an exemplary single strandedlinear library molecule hybridized to an immobilized first surfaceprimer to form a circularized library molecule having an asymmetricallypositioned gap or nick. FIG. 58 (right) is a schematic showing anexemplary single stranded linear library molecule hybridized to animmobilized first surface primer to form a circularized library moleculehaving a symmetrically positioned gap or nick. The schematics shown inFIGS. 57 and 58 represent several embodiments of a circularized librarymolecule comprising a single stranded linear library molecule hybridizedto an immobilized first surface primer.

FIG. 59 is a schematic showing an exemplary covalently closed circularlibrary molecule generated by covalently closing the gap or nick.

FIG. 60 (left) is a schematic showing an exemplary covalently closedcircular library molecule generated by covalently closing the gap ornick. FIG. 60 (right) is a schematic showing an exemplary covalentlyclosed circular library molecule generated by covalently closing the gapor nick. The schematics shown in FIGS. 57 and 58 represent severalembodiments of a covalently closed circular library molecule hybridizedto an immobilized first surface primer.

FIG. 61 is a schematic showing an exemplary on-support rolling circleamplification reaction using a covalently closed circular librarymolecule, the immobilized first surface primer shown in FIG. 55 , and amixture of nucleotides including nucleotides having a scissile moietythat can be cleaved to generate an abasic site. The rolling circleamplification reaction generates an immobilized single stranded nucleicacid concatemer template molecule having at least one nucleotide with ascissile moiety which can be cleaved to generate an abasic site in theimmobilized concatemer template molecule.

FIG. 62 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.61 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers and generates a plurality ofextended forward sequencing primer strands. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 63 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a strand displacing polymerase in the absence ofa soluble primer thereby generating a forward extension strand.

FIG. 64 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble forward sequencing primer therebygenerating a forward extension strand.

FIG. 65 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with a soluble amplification primer therebygenerating a forward extension strand.

FIG. 66 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 63 or 64 .

FIG. 67 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 66 .

FIG. 68 is a schematic showing an exemplary method for generating abasicsites in the immobilized single stranded concatemer template moleculesat the nucleotides having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing concatemertemplate molecules while retaining the plurality of forward extensionstrands and retaining the plurality of immobilized first surfaceprimers. The forward extension strand can be generated by the methoddepicted in FIG. 65 .

FIG. 69 is a schematic showing an exemplary retained forward extensionstrand after removal of the gap-containing concatemer template moleculeas shown in FIG. 68 .

FIG. 70 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 67 .The reverse sequencing reaction can be conducted with a plurality ofsoluble reverse sequencing primers. The retained forward extensionstrand can have two or more extended reverse sequencing primer strandshybridized thereon. The extended reverse sequencing primer strands arenot hybridized to the first surface primer, or covalently joined to thefirst surface primer. Therefore, the extended reverse sequencing primerstrands are not immobilized to the support.

FIG. 71 is a schematic showing an exemplary reverse sequencing reactionconducted on the retained forward extension strand shown in FIG. 69 .The retained forward extension strand can have two or more extendedreverse sequencing primer strands hybridized thereon. The extendedreverse sequencing primer strands are not hybridized to the firstsurface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support.

FIG. 72 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 61 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 73 is a schematic showing an exemplary single stranded nucleic acidconcatemer template molecule immobilized to an immobilized first surfaceprimer. In some embodiments, the immobilized concatemer templatemolecule can be generated by conducting an on-support rolling circleamplification reaction. The arrangement of the various primer bindingsequences is for illustration purposes. The skilled artisan willappreciate that many other arrangements are possible. FIGS. 73-79 showthe workflow of pairwise sequencing the immobilized concatemer templatemolecule depicted in FIG. 73 .

FIG. 74 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.73 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers and generates a plurality ofextended forward sequencing primer strands. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 75 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with soluble amplification primers and stranddisplacing polymerases in the presence of compaction oligonucleotides,thereby generating a forward extension strand and a partially displacedforward extension strand which are hybridized to the immobilizedconcatemer template molecule thereby forming an immobilized amplicon.

FIG. 76 is a schematic showing a continuation of the exemplary stranddisplacing method shown in FIG. 75 , where the polymerase-catalyzedstrand displacing reaction generates a forward extension strand and apartially displaced forward extension strand which are hybridized to theimmobilized concatemer template molecule, and a detached forwardextension strand which is not hybridized to the immobilized concatemertemplate molecule.

FIG. 77 is a schematic showing an exemplary hybridization complexcomprising a forward extension strand and a partially displaced forwardextension strand which are hybridized to the immobilized concatemertemplate molecule, and an immobilized detached forward extension strandwhich is hybridized to the partially displaced forward extension strand.

FIG. 78 is a schematic showing an exemplary reverse sequencing reactionconducted on the hybridization complex shown in FIG. 77 . The reversesequencing reaction can be conducted with a plurality of soluble reversesequencing primers on the partially displaced forward extension strandand the immobilized detached forward extension strand. The reversesequencing reaction generates extended reverse sequencing primerstrands. For the sake of simplicity, FIG. 78 shows one copy of anextended reverse sequencing primer strand on the partially displacedforward extension strand, and one copy of an extended reverse sequencingprimer strand on the immobilized detached forward extension strand. Theskilled artisan will appreciate that the partially displaced forwardextension strand and the immobilized detached forward extension strandcan include two or more extended reverse sequencing primer strandshybridized thereon.

FIG. 79 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 73 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 80 is a schematic showing an exemplary single stranded nucleic acidconcatemer template molecule immobilized to an immobilized first surfaceprimer. In some embodiments, the immobilized concatemer templatemolecule can be generated by conducting an in-solution rolling circleamplification reaction and distributing the rolling circle amplificationreaction onto the support. The arrangement of the various primer bindingsequences is for illustration purposes. The skilled artisan willappreciate that many other arrangements are possible. FIGS. 80-86 showthe workflow of pairwise sequencing the immobilized concatemer templatemolecule depicted in FIG. 80 .

FIG. 81 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.80 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 82 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with soluble amplification primers and stranddisplacing polymerases in the presence of compaction oligonucleotides,thereby generating a forward extension strand and a partially displacedforward extension strand which are hybridized to the immobilizedconcatemer template molecule thereby forming an immobilized amplicon.

FIG. 83 is a schematic showing a continuation of the exemplary stranddisplacing method shown in FIG. 82 , where the polymerase-catalyzedstrand displacing reaction generates a forward extension strand and apartially displaced forward extension strand which are hybridized to theimmobilized concatemer template molecule, and a detached forwardextension strand which is not hybridized to the immobilized concatemertemplate molecule.

FIG. 84 is a schematic showing an exemplary hybridization complexcomprising a forward extension strand and a partially displaced forwardextension strand which are hybridized to the immobilized concatemertemplate molecule, and an immobilized detached forward extension strandwhich is hybridized to the partially displaced forward extension strand.

FIG. 85 is a schematic showing an exemplary reverse sequencing reactionconducted on the hybridization complex shown in FIG. 84 . The reversesequencing reaction can be conducted with a plurality of soluble reversesequencing primers on the partially displaced forward extension strandand the immobilized detached forward extension strand. The reversesequencing reaction generates extended reverse sequencing primerstrands. For the sake of simplicity, FIG. 85 shows one copy of anextended reverse sequencing primer strand on the partially displacedforward extension strand, and one copy of an extended reverse sequencingprimer strand on the immobilized detached forward extension strand. Theskilled artisan will appreciate that the partially displaced forwardextension strand and the immobilized detached forward extension strandcan include two or more extended reverse sequencing primer strandshybridized thereon.

FIG. 86 is a schematic showing an exemplary support having a first andsecond surface primers immobilized thereon. A portion of the immobilizedconcatemer template molecule shown in FIG. 80 is hybridized to theimmobilized second surface primer. The immobilized concatemer templatemolecule has two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer.

FIG. 87 is a schematic showing an exemplary support having a firstsurface primer immobilized thereon, which in some embodiments, can beused to conduct an on-support pairwise sequencing workflow.

FIG. 88 is a schematic showing an exemplary on-support rolling circleamplification reaction using a nucleic acid circular library molecule,the immobilized first surface primer shown in FIG. 87 . The rollingcircle amplification reaction generates an immobilized single strandednucleic acid concatemer template molecule. The arrangement of thevarious primer binding sequences in the nucleic acid circular librarymolecule is for illustration purposes. The skilled artisan willappreciate that many other arrangements are possible. FIGS. 87-94 showthe workflow of pairwise sequencing the immobilized concatemer templatemolecule depicted in FIG. 87 .

FIG. 89 is a schematic showing an exemplary single stranded nucleic acidconcatemer template molecule immobilized to an immobilized first surfaceprimer.

FIG. 90 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.89 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers and generates a plurality ofextended forward sequencing primer strands. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 91 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with soluble amplification primers and stranddisplacing polymerases in the presence of compaction oligonucleotides,thereby generating a forward extension strand and a partially displacedforward extension strand which are hybridized to the immobilizedconcatemer template molecule thereby forming an immobilized amplicon.

FIG. 92 is a schematic showing a continuation of the exemplary stranddisplacing method shown in FIG. 91 , where the polymerase-catalyzedstrand displacing reaction generates a forward extension strand and apartially displaced forward extension strand which are hybridized to theimmobilized concatemer template molecule, and a detached forwardextension strand which is not hybridized to the immobilized concatemertemplate molecule.

FIG. 93 is a schematic showing an exemplary hybridization complexcomprising a forward extension strand and a partially displaced forwardextension strand which are hybridized to the immobilized concatemertemplate molecule, and an immobilized detached forward extension strandwhich is hybridized to the partially displaced forward extension strand.

FIG. 94 is a schematic showing an exemplary reverse sequencing reactionconducted on the hybridization complex shown in FIG. 93 . The reversesequencing reaction can be conducted with a plurality of soluble reversesequencing primers on the partially displaced forward extension strandand the immobilized detached forward extension strand. The reversesequencing reaction generates extended reverse sequencing primerstrands. For the sake of simplicity, FIG. 94 shows one copy of anextended reverse sequencing primer strand on the partially displacedforward extension strand, and one copy of an extended reverse sequencingprimer strand on the immobilized detached forward extension strand. Theskilled artisan will appreciate that the partially displaced forwardextension strand and the immobilized detached forward extension strandcan include two or more extended reverse sequencing primer strandshybridized thereon.

FIG. 95 is a schematic showing an exemplary in-solution rolling circleamplification reaction using a nucleic acid circular library molecule, asoluble first amplification primer, and a mixture of nucleotides. Therolling circle amplification reaction generates in solution singlestranded nucleic acid concatemer molecules. The arrangement of thevarious primer binding sequences in the nucleic acid circular librarymolecule is for illustration purposes. The skilled artisan willappreciate that many other arrangements are possible. FIGS. 95-103 showthe workflow of pairwise sequencing the concatemer molecule depicted inFIG. 96 .

FIG. 96 is a schematic showing an exemplary method comprisingdistributing the rolling circle amplification reaction depicted in FIG.95 onto a support having a first surface primer immobilized thereon. Theconcatemer molecule can hybridize to the immobilized first surfaceprimer.

FIG. 97 is a schematic showing an exemplary method which depicts therolling circle amplification reaction continuing on the support therebygenerating an immobilized concatemer template molecule.

FIG. 98 is a schematic showing an exemplary single stranded nucleic acidconcatemer template molecule immobilized to an immobilized first surfaceprimer.

FIG. 99 is a schematic showing an exemplary forward sequencing reactionconducted on the immobilized concatemer template molecule shown in FIG.98 . The forward sequencing reaction can be conducted with a pluralityof soluble forward sequencing primers. The immobilized concatemertemplate molecule can have two or more extended forward sequencingprimer strands hybridized thereon.

FIG. 100 is a schematic showing an exemplary method for replacing theextended forward sequencing primer strands by conducting a primerextension reaction with soluble amplification primers and stranddisplacing polymerases in the presence of compaction oligonucleotides,thereby generating a forward extension strand and a partially displacedforward extension strand which are hybridized to the immobilizedconcatemer template molecule thereby forming an immobilized amplicon.

FIG. 101 is a schematic showing a continuation of the exemplary stranddisplacing method shown in FIG. 100 , where the polymerase-catalyzedstrand displacing reaction generates a forward extension strand and apartially displaced forward extension strand which are hybridized to theimmobilized concatemer template molecule, and a detached forwardextension strand which is not hybridized to the immobilized concatemertemplate molecule.

FIG. 102 is a schematic showing an exemplary hybridization complexcomprising a forward extension strand and a partially displaced forwardextension strand which are hybridized to the immobilized concatemertemplate molecule, and an immobilized detached forward extension strandwhich is hybridized to the partially displaced forward extension strand.

FIG. 103 is a schematic showing an exemplary reverse sequencing reactionconducted on the hybridization complex shown in FIG. 102 . The reversesequencing reaction can be conducted with a plurality of soluble reversesequencing primers on the partially displaced forward extension strandand the immobilized detached forward extension strand. The reversesequencing reaction generates extended reverse sequencing primerstrands. For the sake of simplicity, FIG. 103 shows one copy of anextended reverse sequencing primer strand on the partially displacedforward extension strand, and one copy of an extended reverse sequencingprimer strand on the immobilized detached forward extension strand. Theskilled artisan will appreciate that the partially displaced forwardextension strand and the immobilized detached forward extension strandcan include two or more extended reverse sequencing primer strandshybridized thereon.

FIG. 104 is a schematic of various exemplary configurations ofmultivalent molecules. Left: schematics of multivalent molecules havinga starburst or helter-skelter configuration. Center: a schematic of amultivalent molecule having a dendrimer configuration. Right: aschematic of multiple multivalent molecules formed by reactingstreptavidin with 4-arm or 8-arm PEG-NETS with biotin and dNTPs.Nucleotide units are designated ‘N’, biotin is designated ‘B’, andstreptavidin is designated ‘SA’.

FIG. 105 is a schematic of an exemplary multivalent molecules comprisinga generic core attached to a plurality of nucleotide-arms.

FIG. 106 is a schematic of an exemplary multivalent molecule comprisinga dendrimer core attached to a plurality of nucleotide-arms.

FIG. 107 shows a schematic of an exemplary multivalent moleculecomprising a core attached to a plurality of nucleotide-arms, where thenucleotide arms comprise biotin, spacer, linker and a nucleotide unit.

FIG. 108 is a schematic of an exemplary nucleotide-arm comprising a coreattachment moiety, spacer, linker and nucleotide unit.

FIG. 109 shows the chemical structure of an exemplary spacer, and thechemical structures of various exemplary linkers, including an 11-atomLinker, 16-atom Linker, 23-atom Linker and an N3 Linker.

FIG. 110 shows the chemical structures of various exemplary linkers,including Linkers 1-9.

FIG. 111 shows the chemical structures of various exemplary linkersjoined/attached to nucleotide units.

FIG. 112 shows the chemical structures of various exemplary linkersjoined/attached to nucleotide units.

FIG. 113 shows the chemical structures of various exemplary linkersjoined/attached to nucleotide units.

FIG. 114 shows the chemical structure of an exemplary nucleotide-arm. Inthis example, the nucleotide unit is connected to the linker via apropargyl amine attachment at the 5 position of a pyrimidine base or the7 position of a purine base. This nucleotide-arm shows an exemplarybiotinylated nucleotide-arm.

FIG. 115 is an exemplary schematic illustration of one embodiment of thelow binding support comprising a glass substrate and alternating layersof hydrophilic coatings which are covalently or non-covalently adheredto the glass, and which further comprises chemically-reactive functionalgroups that serve as attachment sites for oligonucleotide primers (e.g.,capture oligonucleotides and circularization oligonucleotides). In analternative embodiment, the support can be made of any material such asglass, plastic or a polymer material.

FIG. 116A is a schematic of a guanine tetrad (e.g., G-tetrad).

FIG. 116B is a schematic of an intramolecular G-quadruplex structure.

FIG. 117 is a schematic of an exemplary single cycle showing flowing ina nucleic acid relaxing buffer with temperature ramp-up and ramp-down, awashing step, and flowing in a flexing amplification buffer containing astrand-displacing DNA polymerase with temperature ramp-up and MDAincubation and ramp-down. One or more cycles can be conducted of theflowing in a flexing amplification buffer containing a strand-displacingDNA polymerase with temperature ramp-up and MDA amplification andramp-down.

FIG. 118 (left) is a graph showing the error rate from R1 sequencingreads of template molecules having various levels of uracil. FIG. 118(right) is a graph showing the phasing rate from R1 sequencing reads oftemplate molecules having various levels of uracil. The data shows thatsequencing template molecules having lower levels of incorporated uracilyield lower error rates and phasing rates. The level of uracil in thetemplate molecules also affects the intensity ratio of R2/R1 reads.

FIG. 119 is a graph showing increased ratio of signal intensity forR2/R1 sequencing reads when the sequencing workflow employs a cleavingreagent that includes a compound that reduces photo-damage to nucleicacids. Lanes 1, 3, 5 and 7 show the R2/R1 signal intensity usingdifferent cleaving reagent formulations without a compound that reducesphoto-damage. Lanes 2, 4, 6 and 8 show the R2/R1 signal intensity usingcorresponding cleaving reagent formulations that include a compound thatreduces photo-damage.

DETAILED DESCRIPTION Definitions

The headings provided herein are not limitations of the various aspectsof the disclosure, which aspects can be understood by reference to thespecification as a whole.

Unless defined otherwise, technical and scientific terms used hereinhave meanings that are commonly understood by those of ordinary skill inthe art unless defined otherwise. Generally, terminologies pertaining totechniques of molecular biology, nucleic acid chemistry, proteinchemistry, genetics, microbiology, transgenic cell production, andhybridization described herein are those well-known and commonly used inthe art. Techniques and procedures described herein are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the instant specification. For example, seeSambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See alsoAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992). The nomenclatures utilized in connectionwith, and the laboratory procedures and techniques described herein arethose well-known and commonly used in the art.

Unless otherwise required by context herein, singular terms shallinclude pluralities and plural terms shall include the singular.Singular forms “a”, “an” and “the”, and singular use of any word,include plural referents unless expressly and unequivocally limited onone referent.

It is understood the use of the alternative term (e.g., “or”) is takento mean either one or both or any combination thereof of thealternatives.

The term “and/or” used herein is to be taken mean specific disclosure ofeach of the specified features or components with or without the other.For example, the term “and/or” as used in a phrase such as “A and/or B”herein is intended to include: “A and B”; “A or B”; “A” (A alone); and“B” (B alone). In a similar manner, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “Bor C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone);and “C” (C alone).

As used herein and in the appended claims, terms “comprising”,“including”, “having” and “containing”, and their grammatical variants,as used herein are intended to be non-limiting so that one item ormultiple items in a list do not exclude other items that can besubstituted or added to the listed items. It is understood that whereveraspects are described herein with the language “comprising,” otherwiseanalogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

As used herein, the terms “about” and “approximately” refer to a valueor composition that is within an acceptable error range for theparticular value or composition as determined by one of ordinary skillin the art, which will depend in part on how the value or composition ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” or “approximately” can mean within one or more thanone standard deviation per the practice in the art. Alternatively,“about” or “approximately” can mean a range of up to 10% (i.e., ±10%) ormore depending on the limitations of the measurement system. Forexample, about 5 mg can include any number between 4.5 mg and 5.5 mg.Furthermore, particularly with respect to biological systems orprocesses, the terms can mean up to an order of magnitude or up to5-fold of a value. When particular values or compositions are providedin the instant disclosure, unless otherwise stated, the meaning of“about” or “approximately” should be assumed to be within an acceptableerror range for that particular value or composition. Also, where rangesand/or subranges of values are provided, the ranges and/or subranges caninclude the endpoints of the ranges and/or subranges.

The term “biological sample” refers to a single cell, a plurality ofcells, a tissue, an organ, an organism, or section of any of thesebiological samples. The biological sample can be extracted (e.g.,biopsied) from an organism, or obtained from a cell culture grown inliquid or in a culture dish. The biological sample comprises a samplethat is fresh, frozen, fresh frozen, or archived (e.g., formalin-fixedparaffin-embedded; FFPE). The biological sample can be embedded in awax, resin, epoxy or agar. The biological sample can be fixed, forexample in any one or any combination of two or more of acetone,ethanol, methanol, formaldehyde, paraformaldehyde-Triton orglutaraldehyde. The biological sample can be sectioned or non-sectioned.The biological sample can be stained, de-stained or non-stained.

The nucleic acids of interest can be extracted from biological samplesusing any of a number of techniques known to those of skill in the art.For example, a typical DNA extraction procedure comprises (i) collectionof the cell sample or tissue sample from which DNA is to be extracted,(ii) disruption of cell membranes (i.e., cell lysis) to release DNA andother cytoplasmic components, (iii) treatment of the lysed sample with aconcentrated salt solution to precipitate proteins, lipids, and RNA,followed by centrifugation to separate out the precipitated proteins,lipids, and RNA, and (iv) purification of DNA from the supernatant toremove detergents, proteins, salts, or other reagents used during thecell membrane lysis. A variety of suitable commercial nucleic acidextraction and purification kits are consistent with the disclosureherein. Examples include, but are not limited to, the QIAamp kits (forisolation of genomic DNA from human samples) and DNAeasy kits (forisolation of genomic DNA from animal or plant samples) from Qiagen(Germantown, MD), or the Maxwell® and ReliaPrep™ series of kits fromPromega (Madison, WI).

The terms “nucleic acid”, “polynucleotide” and “oligonucleotide” andother related terms used herein are used interchangeably and refer topolymers of nucleotides and are not limited to any particular length.Nucleic acids include recombinant and chemically-synthesized forms.Nucleic acids can be isolated. Nucleic acids include DNA molecules(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of theDNA or RNA generated using nucleotide analogs (e.g., peptide nucleicacids (PNA) and non-naturally occurring nucleotide analogs), andchimeric forms containing DNA and RNA. Nucleic acids can besingle-stranded or double-stranded. Nucleic acids comprise polymers ofnucleotides, where the nucleotides include natural or non-natural basesand/or sugars. Nucleic acids comprise naturally-occurringinternucleosidic linkages, for example phosphdiester linkages. Nucleicacids can lack a phosphate group. Nucleic acids comprise non-naturalinternucleoside linkages, including phosphorothioate, phosphorothiolate,or peptide nucleic acid (PNA) linkages. In some embodiments, nucleicacids comprise a one type of polynucleotides or a mixture of two or moredifferent types of polynucleotides.

The term “universal sequence”, “universal adaptor sequences” and relatedterms refers to a sequence in a nucleic acid molecule that is commonamong two or more polynucleotide molecules. For example, adaptors havingthe same universal sequence can be joined to a plurality ofpolynucleotides so that the population of co-joined molecules carry thesame universal adaptor sequence. Examples of universal adaptor sequencesinclude an amplification primer sequence, a sequencing primer sequenceor a capture primer sequence (e.g., soluble or support-immobilizedcapture primers).

The term “operably linked” and “operably joined” or related terms asused herein refers to juxtaposition of components. The juxtapositionedcomponents can be linked together covalently. For example, two nucleicacid components can be enzymatically ligated together where the linkagethat joins together the two components comprises phosphodiester linkage.A first and second nucleic acid component can be linked together, wherethe first nucleic acid component can confer a function on a secondnucleic acid component. For example, linkage between a primer bindingsequence and a sequence of interest forms a nucleic acid librarymolecule having a portion that can bind to a primer. In another example,a transgene (e.g., a nucleic acid encoding a polypeptide or a nucleicacid sequence of interest) can be ligated to a vector where the linkagepermits expression or functioning of the transgene sequence contained inthe vector. In some embodiments, a transgene is operably linked to ahost cell regulatory sequence (e.g., a promoter sequence) that affectsexpression of the transgene. In some embodiments, the vector comprisesat least one host cell regulatory sequence, including a promotersequence, enhancer, transcription and/or translation initiationsequence, transcription and/or translation termination sequence,polypeptide secretion signal sequences, and the like. In someembodiments, the host cell regulatory sequence controls expression ofthe level, timing and/or location of the transgene.

The terms “linked”, “joined”, “attached”, “appended” and variantsthereof comprise any type of fusion, bond, adherence or associationbetween any combination of compounds or molecules that is of sufficientstability to withstand use in the particular procedure. The procedurecan include but are not limited to: nucleotide binding; nucleotideincorporation; de-blocking (e.g., removal of chain-terminating moiety);washing; removing; flowing; detecting; imaging and/or identifying. Suchlinkage can comprise, for example, covalent, ionic, hydrogen,dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds orassociations involving van der Waals forces, mechanical bonding, and thelike. In some embodiments, such linkage occurs intramolecularly, forexample linking together the ends of a single-stranded ordouble-stranded linear nucleic acid molecule to form a circularmolecule. In some embodiments, such linkage can occur between acombination of different molecules, or between a molecule and anon-molecule, including but not limited to: linkage between a nucleicacid molecule and a solid surface; linkage between a protein and adetectable reporter moiety; linkage between a nucleotide and detectablereporter moiety; and the like. Some examples of linkages can be found,for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition(2008); Aslam, M., Dent, A., “Bioconjugation: Protein CouplingTechniques for the Biomedical Sciences”, London: Macmillan (1998);Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques forthe Biomedical Sciences”, London: Macmillan (1998).

The term “adaptor” and related terms refers to oligonucleotides that canbe operably linked (appended) to a target polynucleotide, where theadaptor confers a function to the co-joined adaptor-target molecule.Adaptors comprise DNA, RNA, chimeric DNA/RNA, or analogs thereof.Adaptors can include at least one ribonucleoside residue. Adaptors canbe single-stranded, double-stranded, or have single-stranded and/ordouble-stranded portions. Adaptors can be configured to be linear,stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length,including 4-100 nucleotides or longer. Adaptors can have blunt ends,overhang ends, or a combination of both. Overhang ends include 5′overhang and 3′ overhang ends. The 5′ end of a single-stranded adaptor,or one strand of a double-stranded adaptor, can have a 5′ phosphategroup or lack a 5′ phosphate group. Adaptors can include a 5′ tail thatdoes not hybridize to a target polynucleotide (e.g., tailed adaptor), oradaptors can be non-tailed. An adaptor can include a sequence that iscomplementary to at least a portion of a primer, such as anamplification primer, a sequencing primer, or a capture primer (e.g.,soluble or immobilized capture primers). Adaptors can include a randomsequence or degenerate sequence. Adaptors can include at least oneinosine residue. Adaptors can include at least one phosphorothioate,phosphorothiolate and/or phosphoramidate linkage. Adaptors can include abarcode sequence which can be used to distinguish polynucleotides (e.g.,insert sequences) from different sample sources in a multiplex assay.Adaptors can include a unique identification sequence (e.g., uniquemolecular index, UMI; or a unique molecular tag) that can be used touniquely identify a nucleic acid molecule to which the adaptor isappended. In some embodiments, a unique identification sequence can beused to increase error correction and accuracy, reduce the rate offalse-positive variant calls and/or increase sensitivity of variantdetection. Adaptors can include at least one restriction enzymerecognition sequence, including any one or any combination of two ormore selected from a group consisting of type I, type II, type III, typeIV, type Hs or type IIB.

The term “nucleic acid template”, “template polynucleotide”, “nucleicacid target” “target polynucleotide”, “template strand” and othervariations refer to a nucleic acid strand that serves as the basisnucleic acid molecule for any of the analysis methods describe herein(e.g., primer extension, amplifying and/or sequencing). The templatenucleic acid can be single-stranded or double-stranded, or the templatenucleic acid can have single-stranded or double-stranded portions. Thetemplate nucleic acid can be obtained from a naturally-occurring source,recombinant form, or chemically synthesized to include any type ofnucleic acid analog. The template nucleic acid can be linear, circular,or other forms. The template nucleic acids can include an insert regionhaving an insert sequence which is also known as a sequence of interest.The template nucleic acids can also include at least one adaptorsequence. The template nucleic acid can be a concatemer having two ortandem copies of a sequence of interest and at least one adaptorsequence. The insert region can be isolated in any form, includingchromosomal, genomic, organellar (e.g., mitochondrial, chloroplast orribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such asprecursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtainedfrom fresh frozen paraffin embedded tissue, needle biopsies, circulatingtumor cells, cell free circulating DNA, or any type of nucleic acidlibrary. The insert region can be isolated from any source includingfrom organisms such as prokaryotes, eukaryotes (e.g., humans, plants andanimals), fungus, viruses cells, tissues, normal or diseased cells ortissues, body fluids including blood, urine, serum, lymph, tumor,saliva, anal and vaginal secretions, amniotic samples, perspiration,semen, environmental samples, culture samples, or synthesized nucleicacid molecules prepared using recombinant molecular biology or chemicalsynthesis methods. The insert region can be isolated from any organ,including head, neck, brain, breast, ovary, cervix, colon, rectum,endometrium, gallbladder, intestines, bladder, prostate, testicles,liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus,skin, heart, larynx, or other organs. The template nucleic acid can besubjected to nucleic acid analysis, including sequencing and compositionanalysis.

The term “polymerase” and its variants, as used herein, comprises anenzyme comprising a domain that binds a nucleotide (or nucleoside) wherethe polymerase can form a complex having a template nucleic acid and acomplementary nucleotide. The polymerase can have one or more activitiesincluding, but not limited to, base analog detection activities, DNApolymerization activity, reverse transcriptase activity, DNA binding,strand displacement activity, and nucleotide binding and recognition. Apolymerase can be any enzyme that can catalyze polymerization ofnucleotides (including analogs thereof) into a nucleic acid strand.Typically but not necessarily such nucleotide polymerization can occurin a template-dependent fashion. Typically, a polymerase comprises oneor more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. In some embodiments, a polymeraseincludes other enzymatic activities, such as for example, 3′ to 5′exonuclease activity or 5′ to 3′ exonuclease activity. In someembodiments, a polymerase has strand displacing activity. A polymerasecan include without limitation naturally occurring polymerases and anysubunits and truncations thereof, mutant polymerases, variantpolymerases, recombinant, fusion or otherwise engineered polymerases,chemically modified polymerases, synthetic molecules or assemblies, andany analogs, derivatives or fragments thereof that retain the ability tocatalyze nucleotide polymerization (e.g., catalytically activefragment). The polymerase includes catalytically inactive polymerases,catalytically active polymerases, reverse transcriptases, and otherenzymes comprising a nucleotide binding domain. In some embodiments, apolymerase can be isolated from a cell, or generated using recombinantDNA technology or chemical synthesis methods. In some embodiments, apolymerase can be expressed in prokaryote, eukaryote, viral, or phageorganisms. In some embodiments, a polymerase can be post-translationallymodified proteins or fragments thereof. A polymerase can be derived froma prokaryote, eukaryote, virus or phage. A polymerase comprisesDNA-directed DNA polymerase and RNA-directed DNA polymerase.

The term “strand displacing” refers to the ability of a polymerase tolocally separate strands of double-stranded nucleic acids and synthesizea new strand in a template-based manner. Strand displacing polymerasesdisplace a complementary strand from a template strand and catalyze newstrand synthesis. Strand displacing polymerases include mesophilic andthermophilic polymerases. Strand displacing polymerases include wildtype enzymes, and variants including exonuclease minus mutants, mutantversions, chimeric enzymes and truncated enzymes. Examples of stranddisplacing polymerases include phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNApolymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, Deep Vent DNA polymerase and KOD DNA polymerase. Thephi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

As used herein, the term “DNA primase-polymerase” and related termsrefers to enzymes having activities of a DNA polymerase and an RNAprimase. A DNA primase-polymerase enzyme can utilize deoxyribonucleotidetriphosphates to synthesize a DNA primer on a single-stranded DNAtemplate in a template-sequence dependent manner, and can extend theprimer strand via nucleotide polymerization (e.g., primer extension), inthe presence of a catalytic divalent cation (e.g., magnesium and/ormanganese). The DNA primase-polymerase include enzymes that are membersof DnaG-like primases (e.g., bacteria) and AEP-like primases (Archaeaand Eukaryotes). An exemplary DNA primase-polymerase enzyme is TthPrimPol from Thermus thermophilus HB27.

As used herein, the term “fidelity” refers to the accuracy of DNApolymerization by template-dependent DNA polymerase. The fidelity of aDNA polymerase is typically measured by the error rate (the frequency ofincorporating an inaccurate nucleotide, i.e., a nucleotide that is notcomplementary to the template nucleotide). The accuracy or fidelity ofDNA polymerization is maintained by both the polymerase activity and the3′-5′ exonuclease activity of a DNA polymerase.

As used herein, the term “binding complex” refers to a complex formed bybinding together a nucleic acid duplex, a polymerase, and a freenucleotide or a nucleotide unit of a multivalent molecule, where thenucleic acid duplex comprises a nucleic acid template moleculehybridized to a nucleic acid primer. In the binding complex, the freenucleotide or nucleotide unit may or may not be bound to the 3′ end ofthe nucleic acid primer at a position that is opposite a complementarynucleotide in the nucleic acid template molecule. A “ternary complex” isan example of a binding complex which is formed by binding together anucleic acid duplex, a polymerase, and a free nucleotide or nucleotideunit of a multivalent molecule, where the free nucleotide or nucleotideunit is bound to the 3′ end of the nucleic acid primer (as part of thenucleic acid duplex) at a position that is opposite a complementarynucleotide in the nucleic acid template molecule.

The term “persistence time” and related terms refers to the length oftime that a binding complex remains stable without dissociation of anyof the components, where the components of the binding complex include anucleic acid template and nucleic acid primer, a polymerase, anucleotide unit of a multivalent molecule or a free (e.g., unconjugated)nucleotide. The nucleotide unit or the free nucleotide can becomplementary or non-complementary to a nucleotide residue in thetemplate molecule. The nucleotide unit or the free nucleotide can bindto the 3′ end of the nucleic acid primer at a position that is oppositea complementary nucleotide residue in the nucleic acid templatemolecule. The persistence time is indicative of the stability of thebinding complex and strength of the binding interactions. Persistencetime can be measured by observing the onset and/or duration of a bindingcomplex, such as by observing a signal from a labeled component of thebinding complex. For example, a labeled nucleotide or a labeled reagentcomprising one or more nucleotides may be present in a binding complex,thus allowing the signal from the label to be detected during thepersistence time of the binding complex. One exemplary label is afluorescent label. The binding complex (e.g., ternary complex) remainsstable until subjected to a condition that causes dissociation ofinteractions between any of the polymerase, template molecule, primerand/or the nucleotide unit or the nucleotide. For example, adissociating condition comprises contacting the binding complex with anyone or any combination of a detergent, EDTA and/or water.

The term “primer” and related terms used herein refers to anoligonucleotide that is capable of hybridizing with a DNA and/or RNApolynucleotide template to form a duplex molecule. Primers comprisenatural nucleotides and/or nucleotide analogs. Primers can berecombinant nucleic acid molecules. Primers may have any length, buttypically range from 4-50 nucleotides. A typical primer comprises a 5′end and 3′ end. The 3′ end of the primer can include a 3′ OH moietywhich serves as a nucleotide polymerization initiation site in apolymerase-catalyzed primer extension reaction. Alternatively, the 3′end of the primer can lack a 3′ OH moiety, or can include a terminal 3′blocking group that inhibits nucleotide polymerization in apolymerase-catalyzed reaction. Any one nucleotide, or more than onenucleotide, along the length of the primer can be labeled with adetectable reporter moiety. A primer can be in solution (e.g., a solubleprimer) or can be immobilized to a support (e.g., a capture primer).

When used in reference to nucleic acid molecules, the terms “hybridize”or “hybridizing” or “hybridization” or other related terms refers tohydrogen bonding between two different nucleic acids to form a duplexnucleic acid. Hybridization also includes hydrogen bonding between twodifferent regions of a single nucleic acid molecule to form aself-hybridizing molecule having a duplex region. Hybridization cancomprise Watson-Crick or Hoogstein binding to form a duplexdouble-stranded nucleic acid, or a double-stranded region within anucleic acid molecule. The double-stranded nucleic acid, or the twodifferent regions of a single nucleic acid, may be wholly complementary,or partially complementary. Complementary nucleic acid strands need nothybridize with each other across their entire length. The complementarybase pairing can be the standard A-T or C-G base pairing, or can beother forms of base-pairing interactions. Duplex nucleic acids caninclude mismatched base-paired nucleotides.

When used in reference to nucleic acids, the terms “extend”,“extending”, “extension” and other variants, refers to incorporation ofone or more nucleotides into a nucleic acid molecule. Nucleotideincorporation comprises polymerization of one or more nucleotides intothe terminal 3′ OH end of a nucleic acid strand (e.g., a nucleic acidprimer), resulting in extension of the nucleic acid strand (e.g.,extended primer). Nucleotide incorporation can be conducted with naturalnucleotides and/or nucleotide analogs. Typically, but not necessarily,nucleotide incorporation occurs in a template-dependent fashion. Anysuitable method of extending a nucleic acid molecule may be used,including primer extension catalyzed by a DNA polymerase or RNApolymerase.

In some embodiments, any of the amplification primer sequences,sequencing primer sequences, capture primer sequences (captureoligonucleotides), target capture sequences, circularization anchorsequences, sample barcode sequences, spatial barcode sequences, oranchor region sequences can be about 3-50 nucleotides in length, orabout 5-40 nucleotides in length, or about 5-25 nucleotides in length.

The term “nucleotides” and related terms refers to a molecule comprisingan aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), andat least one phosphate group. Canonical or non-canonical nucleotides areconsistent with use of the term. The phosphate in some embodimentscomprises a monophosphate, diphosphate, or triphosphate, orcorresponding phosphate analog. The term “nucleoside” refers to amolecule comprising an aromatic base and a sugar. Nucleotides andnucleosides can be non-labeled or labeled with a detectable reportermoiety.

Nucleotides (and nucleosides) typically comprise a hetero cyclic baseincluding substituted or unsubstituted nitrogen-containing parentheteroaromatic ring which are commonly found in nucleic acids, includingnaturally-occurring, substituted, modified, or engineered variants, oranalogs of the same. The base of a nucleotide (or nucleoside) is capableof forming Watson-Crick and/or Hoogstein hydrogen bonds with anappropriate complementary base. Exemplary bases include, but are notlimited to, purines and pyrimidines such as: 2-aminopurine,2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-Δ²-isopentenyladenine(6iA), N⁶-Δ²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶-methyladenine,guanine (G), isoguanine, N²-dimethylguanine (dmG), 7-methylguanine(7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine andO⁶-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C),5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT),5,6-dihydrothymine, O⁴-methylthymine, uracil (U), 4-thiouracil (4sU) and5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines;hydroxymethylcytosines; 5-methycytosines; base (Y); as well asmethylated, glycosylated, and acylated base moieties; and the like.Additional exemplary bases can be found in Fasman, 1989, in “PracticalHandbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press,Boca Raton, Fla.

Nucleotides (and nucleosides) typically comprise a sugar moiety, such ascarbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48),acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal ChemistryLetters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al.,1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No.5,558,991). The sugar moiety comprises: ribosyl; 2′-deoxyribosyl;3′-deoxyribosyl; 2′,3′-dideoxyribosyl; 2′,3′-didehydrodideoxyribosyl;2′-alkoxyribosyl; 2′-azidoribosyl; 2′-aminoribosyl; 2′-fluororibosyl;2′-mercaptoriboxyl; 2′-alkylthioribosyl; 3′-alkoxyribosyl;3′-azidoribosyl; 3′-aminoribosyl; 3′-fluororibosyl; 3′-mercaptoriboxyl;3′-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

In some embodiments, nucleotides comprise a chain of one, two or threephosphorus atoms where the chain is typically attached to the 5′ carbonof the sugar moiety via an ester or phosphoramide linkage. In someembodiments, the nucleotide is an analog having a phosphorus chain inwhich the phosphorus atoms are linked together with intervening O, S,NH, methylene or ethylene. In some embodiments, the phosphorus atoms inthe chain include substituted side groups including O, S or BH₃. In someembodiments, the chain includes phosphate groups substituted withanalogs including phosphoramidate, phosphorothioate, phosphordithioate,and O-methylphosphoroamidite groups.

The term “reporter moiety”, “reporter moieties” or related terms refersto a compound that generates, or causes to generate, a detectablesignal. A reporter moiety is sometimes called a “label”. Any suitablereporter moiety may be used, including luminescent, photoluminescent,electroluminescent, bioluminescent, chemiluminescent, fluorescent,phosphorescent, chromophore, radioisotope, electrochemical, massspectrometry, Raman, hapten, affinity tag, atom, or an enzyme. Areporter moiety generates a detectable signal resulting from a chemicalor physical change (e.g., heat, light, electrical, pH, saltconcentration, enzymatic activity, or proximity events). A proximityevent includes two reporter moieties approaching each other, orassociating with each other, or binding each other. It is well known toone skilled in the art to select reporter moieties so that each absorbsexcitation radiation and/or emits fluorescence at a wavelengthdistinguishable from the other reporter moieties to permit monitoringthe presence of different reporter moieties in the same reaction or indifferent reactions. Two or more different reporter moieties can beselected having spectrally distinct emission profiles, or having minimaloverlapping spectral emission profiles. Reporter moieties can be linked(e.g., operably linked) to nucleotides, nucleosides, nucleic acids,enzymes (e.g., polymerases or reverse transcriptases), or support (e.g.,surfaces).

A reporter moiety (or label) comprises a fluorescent label or afluorophore. Exemplary fluorescent moieties which may serve asfluorescent labels or fluorophores include, but are not limited tofluorescein and fluorescein derivatives such as carboxyfluorescein,tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein,fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein,fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide,carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine andrhodamine derivatives such as TRITC, TMR, lissamine rhodamine, TexasRed, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine,TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissaminerhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Redhydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS,AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivativessuch as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY530/550 C3 hydrazide, BODIPY 493/503 C3 hydrazide, BODIPY FL C3hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, CascadeBlue and derivatives such as Cascade Blue acetyl azide, Cascade Bluecadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide,Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide,Lucifer Yellow CH, cyanine and derivatives such as indolium basedcyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyaninedyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes,imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates andderivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates,Terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCyclerRed dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Greendyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes,Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes andothers known in the art such as those described in Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), orHermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof,or any combination thereof. Cyanine dyes may exist in either sulfonatedor non-sulfonated forms, and consist of two indolenin, benzo-indolium,pyridium, thiozolium, and/or quinolinium groups separated by apolymethine bridge between two nitrogen atoms. Commercially availablecyanine fluorophores include, for example, Cy3, (which may comprise1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indoliumor1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate),Cy5 (which may comprise1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-iumor1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate),and Cy7 (which may comprise1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indoliumor1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate),where “Cy” stands for ‘cyanine’, and the first digit identifies thenumber of carbon atoms between two indolenine groups. Cy2 which is anoxazole derivative rather than indolenin, and the benzo-derivatizedCy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

In some embodiments, the reporter moiety can be a FRET pair, such thatmultiple classifications can be performed under a single excitation andimaging step. As used herein, FRET may comprise excitation exchange(Forster) transfers, or electron-exchange (Dexter) transfers.

The term “support” as used herein refers to a substrate that is designedfor deposition of biological molecules or biological samples for assaysand/or analyses. Examples of biological molecules to be deposited onto asupport include nucleic acids (e.g., DNA, RNA), polypeptides,saccharides, lipids, a single cell or multiple cells. Examples ofbiological samples include but are not limited to saliva, phlegm, mucus,blood, plasma, serum, urine, stool, sweat, tears and fluids from tissuesor organs.

In some embodiments, the support is solid, semi-solid, or a combinationof both. In some embodiments, the support is porous, semi-porous,non-porous, or any combination of porosity. In some embodiments, thesupport can be substantially planar, concave, convex, or any combinationthereof. In some embodiments, the support can be cylindrical, forexample comprising a capillary or interior surface of a capillary.

In some embodiments, the surface of the support can be substantiallysmooth. In some embodiments, the support can be regularly or irregularlytextured, including bumps, etched, pores, three-dimensional scaffolds,or any combination thereof.

In some embodiments, the support comprises a bead having any shape,including spherical, hemi-spherical, cylindrical, barrel-shaped,toroidal, disc-shaped, rod-like, conical, triangular, cubical,polygonal, tubular or wire-like.

The support can be fabricated from any material, including but notlimited to glass, fused-silica, silicon, a polymer (e.g., polystyrene(PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA),polycarbonate (PC), polypropylene (PP), polyethylene (PE), high densitypolyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefincopolymers (COC), polyethylene terephthalate (PET)), or any combinationthereof. Various compositions of both glass and plastic substrates arecontemplated.

The support can have a plurality (e.g., two or more) of nucleic acidtemplates immobilized thereon. The plurality of immobilized nucleic acidtemplates have the same sequence or have different sequences. In someembodiments, individual nucleic acid template molecules in the pluralityof nucleic acid templates are immobilized to a different site on thesupport. In some embodiments, two or more individual nucleic acidtemplate molecules in the plurality of nucleic acid templates areimmobilized to a site on the support.

The term “array” refers to a support comprising a plurality of siteslocated at pre-determined locations on the support to form an array ofsites. The sites can be discrete and separated by interstitial regions.In some embodiments, the pre-determined sites on the support can bearranged in one dimension in a row or a column, or arranged in twodimensions in rows and columns. In some embodiments, the plurality ofpre-determined sites is arranged on the support in an organized fashion.In some embodiments, the plurality of pre-determined sites is arrangedin any organized pattern, including rectilinear, hexagonal patterns,grid patterns, patterns having reflective symmetry, patterns havingrotational symmetry, or the like. The pitch between different pairs ofsites can be that same or can vary. In some embodiments, the supportcomprises at least 10² sites, at least 10³ sites, at least 10⁴ sites, atleast 10⁵ sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸sites, at least 10⁹ sites, at least 10¹⁰ sites, at least 10¹¹ sites, atleast 10¹² sites, at least 10¹³ sites, at least 10¹⁴ sites, at least10¹⁵ sites, or more, where the sites are located at pre-determinedlocations on the support. In some embodiments, a plurality ofpre-determined sites on the support (e.g., 10²-10¹⁵ sites or more) areimmobilized with nucleic acid templates to form a nucleic acid templatearray. In some embodiments, the nucleic acid templates that areimmobilized at a plurality of pre-determined sites by hybridization toimmobilized surface capture primers, or the nucleic acid templates arecovalently attached to the surface capture primer. In some embodiments,the nucleic acid templates that are immobilized at a plurality ofpre-determined sites, for example immobilized at 10²-10¹⁵ sites or more.In some embodiments, the immobilized nucleic acid templates areclonally-amplified to generate immobilized nucleic acid polonies at theplurality of pre-determined sites. In some embodiments, individualimmobilized nucleic acid polonies comprise single-stranded ordouble-stranded concatemers.

In some embodiments, a support comprising a plurality of sites locatedat random locations on the support is referred to herein as a supporthaving randomly located sites thereon. The location of the randomlylocated sites on the support are not pre-determined. The plurality ofrandomly-located sites is arranged on the support in a disordered and/orunpredictable fashion. In some embodiments, the support comprises atleast 10² sites, at least 10³ sites, at least 10⁴ sites, at least 10⁵sites, at least 10⁶ sites, at least 10⁷ sites, at least 10⁸ sites, atleast 10⁹ sites, at least 10¹⁰ sites, at least 10¹¹ sites, at least 10¹²sites, at least 10¹³ sites, at least 10¹⁴ sites, at least 10¹⁵ sites, ormore, where the sites are randomly located on the support. In someembodiments, a plurality of randomly located sites on the support (e.g.,10²-10¹⁵ sites or more) are immobilized with nucleic acid templates toform a support immobilized with nucleic acid templates. In someembodiments, the nucleic acid templates that are immobilized at aplurality of randomly located sites by hybridization to immobilizedsurface capture primers, or the nucleic acid templates are covalentlyattached to the surface capture primer. In some embodiments, the nucleicacid templates that are immobilized at a plurality of randomly locatedsites, for example immobilized at 10²-10¹⁵ sites or more. In someembodiments, the immobilized nucleic acid templates areclonally-amplified to generate immobilized nucleic acid polonies at theplurality of randomly located sites. In some embodiments, individualimmobilized nucleic acid polonies comprise single-stranded ordouble-stranded concatemers.

When used in reference to a low binding surface coating, one or morelayers of a multi-layered surface coating may comprise a branchedpolymer or may be linear. Examples of suitable branched polymersinclude, but are not limited to, branched PEG, branched poly(vinylalcohol) (branched PVA), branched poly(vinyl pyridine), branchedpoly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid)(branched PAA), branched polyacrylamide, branchedpoly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methylmethacrylate) (branched PMA), branched poly(2-hydroxylethylmethacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol)methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid(branched PGA), branched poly-lysine, branched poly-glucoside, anddextran.

In some embodiments, the branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein maycomprise at least 4 branches, at least 5 branches, at least 6 branches,at least 7 branches, at least 8 branches, at least 9 branches, at least10 branches, at least 12 branches, at least 14 branches, at least 16branches, at least 18 branches, at least 20 branches, at least 22branches, at least 24 branches, at least 26 branches, at least 28branches, at least 30 branches, at least 32 branches, at least 34branches, at least 36 branches, at least 38 branches, or at least 40branched.

Linear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may have amolecular weight of at least 500, at least 1,000, at least 2,000, atleast 3,000, at least 4,000, at least 5,000, at least 10,000, at least15,000, at least 20,000, at least 25,000, at least 30,000, at least35,000, at least 40,000, at least 45,000, or at least 50,000 daltons.

In some embodiments, e.g., wherein at least one layer of a multi-layeredsurface comprises a branched polymer, the number of covalent bondsbetween a branched polymer molecule of the layer being deposited andmolecules of the previous layer may range from about one covalentlinkage per molecule and about 32 covalent linkages per molecule. Insome embodiments, the number of covalent bonds between a branchedpolymer molecule of the new layer and molecules of the previous layermay be at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 12,at least 14, at least 16, at least 18, at least 20, at least 22, atleast 24, at least 26, at least 28, at least 30, or at least 32 covalentlinkages per molecule.

Any reactive functional groups that remain following the coupling of amaterial layer to the surface may optionally be blocked by coupling asmall, inert molecule using a high yield coupling chemistry. Forexample, in the case that amine coupling chemistry is used to attach anew material layer to the previous one, any residual amine groups maysubsequently be acetylated or deactivated by coupling with a small aminoacid such as glycine.

The number of layers of low non-specific binding material, e.g., ahydrophilic polymer material, deposited on the surface, may range from 1to about 10. In some embodiments, the number of layers is at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10. In some embodiments, the number oflayers may be at most 10, at most 9, at most 8, at most 7, at most 6, atmost 5, at most 4, at most 3, at most 2, or at most 1. Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someembodiments the number of layers may range from about 2 to about 4. Insome embodiments, all of the layers may comprise the same material. Insome embodiments, each layer may comprise a different material. In someembodiments, the plurality of layers may comprise a plurality ofmaterials. In some embodiments at least one layer may comprise abranched polymer. In some embodiment, all of the layers may comprise abranched polymer.

One or more layers of low non-specific binding material may in somecases be deposited on and/or conjugated to the substrate surface using apolar protic solvent, a polar or polar aprotic solvent, a nonpolarsolvent, or any combination thereof. In some embodiments the solventused for layer deposition and/or coupling may comprise an alcohol (e.g.,methanol, ethanol, propanol, etc.), another organic solvent (e.g.,acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),etc.), water, an aqueous buffer solution (e.g., phosphate buffer,phosphate buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS),etc.), or any combination thereof. In some embodiments, an organiccomponent of the solvent mixture used may comprise at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% of the total, with the balance made up of water oran aqueous buffer solution. In some embodiments, an aqueous component ofthe solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% of the total, with the balance made up of an organicsolvent. The pH of the solvent mixture used may be less than 6, about 6,6.5, 7, 7.5, 8, 8.5, 9, or greater than pH 9.

The term “branched polymer” and related terms refers to a polymer havinga plurality of functional groups that help conjugate a biologicallyactive molecule such as a nucleotide, and the functional group can beeither on the side chain of the polymer or directly attaches to acentral core or central backbone of the polymer. The branched polymercan have linear backbone with one or more functional groups coming offthe backbone for conjugation. The branched polymer can also be a polymerhaving one or more sidechains, wherein the side chain has a sitesuitable for conjugation. Examples of the functional group include butare limited to hydroxyl, ester, amine, carbonate, acetal, aldehyde,aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activesulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate,isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine,iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, andtresylate.

When used in reference to immobilized nucleic acids, the term“immobilized” and related terms refer to nucleic acid molecules that areattached to a support through covalent bond or non-covalent interaction,or attached to a coating on the support, or buried within a matrixformed by a coating on the support, where the nucleic acid moleculesinclude surface capture primers, nucleic acid template molecules andextension products of capture primers. Extension products of captureprimers includes nucleic acid concatemers that can form nucleic acidpolonies.

In some embodiments, one or more nucleic acid templates are immobilizedon the support, for example immobilized at the sites on the support. Insome embodiments, the one or more nucleic acid templates areclonally-amplified. In some embodiments, the one or more nucleic acidtemplates are clonally-amplified off the support (e.g., in-solution) andthen deposited onto the support and immobilized on the support. In someembodiments, the clonal amplification reaction of the one or morenucleic acid templates is conducted on the support resulting inimmobilization on the support. In some embodiments, the one or morenucleic acid templates are clonally-amplified (e.g., in solution or onthe support) using a nucleic acid amplification reaction, including anyone or any combination of: polymerase chain reaction (PCR), multipledisplacement amplification (MDA), transcription-mediated amplification(TMA), nucleic acid sequence-based amplification (NASBA), stranddisplacement amplification (SDA), real-time SDA, bridge amplification,isothermal bridge amplification, rolling circle amplification (RCA),circle-to-circle amplification, helicase-dependent amplification,recombinase-dependent amplification, and/or single-stranded binding(SSB) protein-dependent amplification.

The term “surface primer”, “surface capture primer” and related termsrefers to single-stranded oligonucleotides that are immobilized to asupport and comprise a sequence that can hybridize to at least a portionof a nucleic acid template molecule. Surface primers can be used toimmobilize template molecules to a support via hybridization. Surfaceprimers can be immobilized to a support in a manner that resists primerremoval during flowing, washing, aspirating, and changes in temperature,pH, salts, chemical and/or enzymatic conditions. Typically, but notnecessarily, the 5′ end of a surface primer can be immobilized to asupport. Alternatively, an interior portion or the 3′ end of a surfaceprimer can be immobilized to a support.

The surface primers comprise DNA, RNA, or analogs thereof. The surfaceprimers can include a combination of DNA and RNA. The sequence ofsurface primers can be wholly complementary or partially complementaryalong their length to at least a portion of the nucleic acid templatemolecule (e.g., linear or circular template molecules). A support caninclude a plurality of immobilized surface primers having the samesequence, or having two or more different sequences. Surface primers canbe any length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

A surface primer can include a terminal 3′ nucleotide having a sugar 3′OH moiety which is extendible for nucleotide polymerization (e.g.,polymerase catalyzed polymerization). A surface primer can include aterminal 3′ nucleotide having a moiety that blocks polymerase-catalyzedextension. A surface primer can include a terminal 3′ nucleotide havingthe 3′ sugar position linked to a chain-terminating moiety that inhibitsnucleotide polymerization. The 3′ chain-terminating moiety can beremoved (e.g., de-blocked) to convert the 3′ end to an extendible 3′ OHend using a de-blocking agent. Examples of chain terminating moietiesinclude alkyl group, alkenyl group, alkynyl group, allyl group, arylgroup, benzyl group, azide group, amine group, amide group, keto group,isocyanate group, phosphate group, thio group, disulfide group,carbonate group, urea group, or silyl group. Azide type chainterminating moieties including azide, azido and azidomethyl groups.Examples of de-blocking agents include a phosphine compound, such asTris(2-carboxyethyl)phosphine (TCEP) and bis-sulfo triphenyl phosphine(BS-TPP), for chain-terminating groups azide, azido and azidomethylgroups. Examples of de-blocking agents includetetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) with piperidine, orwith 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), forchain-terminating groups alkyl, alkenyl, alkynyl and allyl. Examples ofa de-blocking agent includes Pd/C for chain-terminating groups aryl andbenzyl. Examples of de-blocking agents include phosphine,beta-mercaptoethanol or dithiothritol (DTT), for chain-terminatinggroups amine, amide, keto, isocyanate, phosphate, thio and disulfide.Examples of de-blocking agents include potassium carbonate (K₂CO₃) inMeOH, triethylamine in pyridine, and Zn in acetic acid (AcOH), forcarbonate chain-terminating groups. Examples of de-blocking agentsinclude tetrabutylammonium fluoride, pyridine-HF, with ammoniumfluoride, and triethylamine trihydrofluoride, for chain-terminatinggroups urea and silyl.

In some embodiment, the plurality of immobilized surface capture primerson the support are in fluid communication with each other to permitflowing a solution of reagents (e.g., linear or circular nucleic acidtemplate molecules, soluble primers, enzymes, nucleotides, divalentcations, buffers, reagents and the like) onto the support so that theplurality of immobilized surface capture primers on the support can beessentially simultaneously reacted with the reagents in a massivelyparallel manner. In some embodiments, the fluid communication of theplurality of immobilized surface capture primers can be used to conductnucleic acid amplification reactions (e.g., RCA, MDA, PCR and bridgeamplification) essentially simultaneously on the plurality ofimmobilized surface capture primers.

In some embodiment, the plurality of immobilized single stranded nucleicacid concatemer template molecules on the support are in fluidcommunication with each other to permit flowing a solution of reagents(e.g., soluble primers, enzymes, nucleotides, divalent cations, buffers,reagents and the like) onto the support so that the plurality ofimmobilized concatemer template molecules on the support can beessentially simultaneously reacted with the reagents in a massivelyparallel manner. In some embodiments, the fluid communication of theplurality of immobilized single stranded nucleic acid concatemertemplate molecules can be used to conduct nucleotide binding assaysand/or conduct nucleotide polymerization reactions (e.g., primerextension or sequencing) essentially simultaneously on the plurality ofimmobilized single stranded nucleic acid concatemer template molecules,and optionally to conduct detection and imaging for massively parallelsequencing.

When used in reference to nucleic acids, the terms “amplify”,“amplifying”, “amplification”, and other related terms include producingmultiple copies of an original polynucleotide template molecule, wherethe copies comprise a sequence that is complementary to the templatesequence, or the copies comprise a sequence that is the same as thetemplate sequence. In some embodiments, the copies comprise a sequencethat is substantially identical to a template sequence, or issubstantially identical to a sequence that is complementary to thetemplate sequence.

The present disclosure provides various pH buffering agents. The fullname of the pH buffering agents is listed herein.

The term “Tris” refers to a pH buffering agentTris(hydroxymethyl)-aminomethane. The term “Tris-HCl” refers to a pHbuffering agent Tris(hydroxymethyl)-aminomethane hydrochloride. The term“Tricine” refers to a pH buffering agent N-[tris(hydroxymethyl)methyl]glycine. The term “Bicine” refers to a pH buffering agentN,N-bis(2-hydroxyethyl)glycine. The term “Bis-Tris propane” refers to apH buffering agent 1,3 Bis[tris(hydroxymethyl)methylamino]propane. Theterm “HEPES” refers to a pH buffering agent4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The term “MES”refers to a pH buffering agent 2-(N-morpholino)ethanesulfonic acid). Theterm “MOPS” refers to a pH buffering agent3-(N-morpholino)propanesulfonic acid. The term “MOPSO” refers to a pHbuffering agent 3-(N-morpholino)-2-hydroxypropanesulfonic acid. The term“BES” refers to a pH buffering agentN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid. The term “TES”refers to a pH buffering agent 2-[(2-Hydroxy-1,1bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid). The term “CAPS” refers to a pHbuffering agent 3-(cyclohexylamino)-1-propanesuhinic acid. The term“TAPS” refers to a pH buffering agentN-[Tris(hydroxymethyl)methyl]-3-amino propane sulfonic acid. The term“TAPSO” refers to a pH buffering agentN-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropansulfonic acid. Theterm “ACES” refers to a pH buffering agentN-(2-Acetamido)-2-aminoethanesulfonic acid. The term “PIPES” refers to apH buffering agent piperazine-1,4-bis(2-ethanesulfonic acid.

INTRODUCTION

The present disclosure provides compositions and methods that employ thecompositions for conducting pairwise sequencing and for generatingconcatemer template molecules for pairwise sequencing.

Pairwise sequencing comprises obtaining a first sequencing read of afirst region of a first nucleic acid strand (e.g., sense strand), andobtaining a second sequencing read of a second region of a secondnucleic acid strand that is complementary to the first stand (e.g.,anti-sense strand), wherein the first and second strands correspond totwo complementary strands of the same double stranded template molecule.The first sequencing read of the first sequenced region and the secondsequencing read of the second sequenced region can having overlappingsequences which correspond to complementary sequences from the first andsecond strands of the double stranded template molecule. The first andsecond sequencing reads can be aligned so that the overlappingsequencing reads can yield sequence information of a paired region inthe original double stranded nucleic acid source (e.g., a paired regionin the genome), and the accuracy of the sequence information can beascertained from the first and second sequencing reads with a high levelof confidence. The first sequencing read of the first sequenced regionand the second sequencing read of the second sequenced region do notnecessarily have overlapping sequences in which case sequenceinformation of a paired region in the original double stranded nucleicacid source cannot be ascertained with a high level of confidence. Thefirst and second sequencing reads can initiate at one end of theirrespective template molecules, or can initiate at an internal position.

The compositions and methods for pairwise sequencing described hereinoffers several advantages which improves the quality of the sequencingdata, including increased signal intensity which improves base callaccuracy. The pairwise sequencing methods also saves time by obviatingthe need to prepare separate nucleic acid libraries each correspondingto the sense and anti-sense strands of the double stranded templatemolecule having the sequence of interest. Additionally, the pairwisesequencing methods generate and sequence the sense and anti-sensestrands directly on the support/substrate used to conduct the sequencingreactions.

The present disclosure provides pairwise sequencing methods that employa support having a plurality of surface primers immobilized thereon. Theimmobilized surface primers are in fluid communication with each otherto permit flowing various solutions of linear or circular nucleic acidtemplate molecules, soluble primers, enzymes, nucleotides, divalentcations, buffers, reagents, and the like, onto the support so that theplurality of immobilized surface primers (and products generated fromthe immobilized surface primers) react with the solutions in a massivelyparallel manner.

The present disclosure provides pairwise sequencing methods comprisingthe steps: (a) providing a plurality of single stranded nucleic acidconcatemer template molecules immobilized to a support; (b) sequencingthe plurality of immobilized concatemer template molecules with a firstplurality of sequencing polymerases, a plurality of soluble forwardsequencing primers and a first plurality of multivalent molecules,thereby generating a plurality of extended forward sequencing primerstrands; (c) retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized concatemer template molecules byconducting a primer extension reaction; (d) removing the retainedimmobilized concatemer template molecules while retaining the pluralityof forward extension strands; and (e) sequencing the plurality ofretained forward extension strands with a second plurality of sequencingpolymerases, a plurality of soluble reverse sequencing primers and asecond plurality of multivalent molecules. In some embodiments,individual concatemer template molecules in the plurality areimmobilized to a surface primer where the surface primer is immobilizedto the support. In some embodiments, individual concatemer templatemolecules are covalently joined to a surface primer, or individualconcatemer template molecules are hybridized to a surface primer. Insome embodiments, the immobilized surface primer includes or lacks anucleotide having a scissile moiety that can be cleaved to generate anabasic site in the surface primer. In some embodiments, the plurality ofconcatemer template molecules comprise at least one nucleotide having ascissile moiety that can be cleaved to generate an abasic site in theconcatemer template molecule. In some embodiments, the plurality ofconcatemer template molecules lack a nucleotide having a scissilemoiety. Exemplary nucleotides having a scissile moiety (e.g., in thesurface primer or the concatemer template molecule) include uridine,8-oxo-7,8-dihydrogunine and deoxyinosine.

In some embodiments, pairwise sequencing methods include a rollingcircle amplification reaction which is conducted on-support bydistributing a plurality of single stranded circular library moleculesonto the support having a plurality of surface primers immobilizedthereon. Individual surface primers are designed to capture, viahybridization, a single circular library molecule. The rolling circleamplification reaction can be conducted on the support. In someembodiments, for the on-support RCA reaction, a solution of singlestranded circular library molecules is flowed onto the support so thatindividual circular molecules are captured via hybridization toindividual surface primers. Individual circular library moleculesinclude at least a sequence of interest and a universal surface primerbinding site, and optionally include universal sequencing primer bindingsites, universal amplification primer binding site, an additionalsurface primer binding site, and a sample barcode and/or a molecularindex. A single immobilized surface primer will capture a singlecircular library molecule and the rolling circle amplification reactiongenerates a single stranded linear concatemer that is covalently linkedto the immobilized surface primer by employing the terminal 3′ end ofthe surface primer as a primer extension initiation site. Thus,individual concatemer molecules are immobilized to the support asconcatemers that are covalently linked to an immobilized surface primer.The single stranded concatemer includes multiple tandem copies of thesequence of interest and the universal sequencing primer binding sites.A single surface primer will capture a single circular library moleculeand generate a single concatemer molecule.

In some embodiments, pairwise sequencing methods include a rollingcircle amplification reaction which is conducted in-solution to generatea plurality of concatemers which are distributed onto the support havinga plurality of surface primers immobilized thereon. Individual surfaceprimers are designed to capture, via hybridization, a single concatemerhaving complementary sequences of the circular library molecules. Therolling circle amplification reaction can continue on the support. Insome embodiments, for the in-solution RCA reaction, a plurality ofsingle stranded circular library molecules are subjected to a rollingcircle amplification reaction in a reaction vessel. Individual circularlibrary molecules include at least a sequence of interest and auniversal surface primer binding site, and optionally include universalsequencing primer binding sites, universal amplification primer bindingsite, an additional surface primer binding site, and a sample barcodeand/or a molecular index. The RCA reaction can be conducted for a veryshort period of time or can be conducted for longer periods of time, togenerate a plurality of concatemers hybridized to their respectivecircular library molecules which are then distributed onto the supporthaving a plurality of surface primers immobilized thereon. A solution ofconcatemer molecules is flowed onto the support so that individualconcatemer molecules are captured via hybridization to individualsurface primers. Individual concatemer molecules include at least asequence of interest, universal surface primer binding site(s),universal sequencing primer binding sites, and optionally a samplebarcode and/or a molecular index. A single immobilized surface primerwill capture a single concatemer molecule and the rolling circleamplification reaction (now on the support) continues thereby extendingthe single stranded concatemer that is hybridized to the immobilizedsurface primer. Thus, individual concatemer molecules are immobilized tothe support as concatemers that are hybridized to an immobilized surfaceprimer. The single stranded concatemer includes multiple tandem copiesof the sequence of interest and the universal sequencing primer bindingsites. A single surface primer will capture a single concatemer moleculeand generate a single extended concatemer molecule.

The rolling circle amplification reaction, conducted either byin-solution or on-support, will generate concatemers that areimmobilized to the support. Immobilized concatemers offer severaladvantages compared to non-concatemer molecules. The number of tandemcopies in the concatemer is tunable by controlling the time, temperatureand concentration of reagents of the in-solution or on-support rollingcircle amplification reaction. The concatemer can self-collapse into acompact nucleic acid nanoball. Inclusion of one or more compactionoligonucleotides during the RCA reaction can further compact the sizeand/or shape of the nanoball. An increase in the number of tandem copiesin a given concatemer increases the number of sites along the concatemerfor hybridizing to multiple sequencing primers which serve as multipleinitiation sites for polymerase-catalyzed sequencing reactions. When thesequencing reaction employs detectably labeled nucleotides and/ordetectably labeled multivalent molecules (e.g., having nucleotideunits), the signals emitted by the nucleotides or nucleotide units thatparticipate in the parallel sequencing reactions along the concatemeryields an increased signal intensity for each concatemer. Multipleportions of a given concatemer can be simultaneously sequenced.Furthermore, a plurality of binding complexes can form along aparticular concatemer molecule, each binding complex comprising asequencing polymerase bound to a multivalent molecule wherein theplurality of binding complexes remain stable without dissociationresulting in increased persistence time which increases signal intensityand reduces imaging time.

The level of sequencing accuracy can be further improved by obtainingpartially or wholly overlapping sequencing reads from both sense andanti-sense strands, and aligning the sequencing reads which providesredundant sequencing data.

Thus, the pairwise sequencing compositions and methods described hereinprovide improved sequencing data quality in a massively parallel manner.

Methods for Pairwise Sequencing—Generating Abasic Sites

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a plurality of immobilized single stranded nucleicacid concatemer template molecules each comprising at least onenucleotide having a scissile moiety, wherein individual concatemertemplate molecules in the plurality are immobilized to a first surfaceprimer that is immobilized to a support, and wherein the immobilizedfirst surface primer lacks a nucleotide having a scissile moiety. Insome embodiments, the support comprises a plurality of first surfaceprimers. In some embodiments, the support lacks a plurality of secondsurface primers. In some embodiments, the support comprises a pluralityof first and second surface primers.

In some embodiments, individual immobilized concatemer templatemolecules are covalently joined to an immobilized surface primer (e.g.,an immobilized first surface primer) (FIG. 1 ). In an alternativeembodiment, individual immobilized concatemer template molecules arehybridized to an immobilized surface primer (e.g., an immobilized firstsurface primer) (FIG. 13 ).

In some embodiments, individual concatemer template molecules in theplurality comprise two or more copies of a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of: (i) two or morecopies of a universal binding sequence for a soluble forward sequencingprimer, (ii) two or more copies of a universal binding sequence for asoluble reverse sequencing primer, (iii) two or more copies of auniversal binding sequence for an immobilized first surface primer, (iv)two or more copies of a universal binding sequence for an immobilizedsecond surface primer, (v) two or more copies of a universal bindingsequence for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence for a second solubleamplification primer, (vii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the scissile moiety in the immobilized concatemertemplate molecules of step (a) can be converted into abasic sites in theimmobilized concatemer template molecules. In some embodiments, thescissile moiety in the immobilized concatemer template moleculescomprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine. In the concatemer template molecules, the uridine can beconverted to an abasic site using uracil DNA glycosylase (UDG), the8oxoG can be converted to an abasic site using FPG glycosylase, and thedeoxyinosine can be converted to an abasic site using AlkA glycosylase.In some embodiments, the immobilized concatemer template moleculesinclude 1-20, 20-40, 40-60, 60-80, 80-100, or a higher number ofnucleotides with a scissile moiety. In some embodiments, about 0.1-1%,or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30% or ahigher percent of the dTTP in the immobilized concatemer templatemolecules are replaced with nucleotides having a scissile moiety. Insome embodiments, the nucleotides having a scissile moiety aredistributed at random positions along individual immobilized concatemertemplate molecules. In some embodiments, the nucleotides having ascissile moiety are distributed at different positions in the differentimmobilized concatemer template molecules.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized first surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedfirst surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized first surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a 3′ non-extendible moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the immobilized concatemer template moleculesfurther comprise two or more copies of a universal binding sequence (orcomplementary sequence thereof) for an immobilized second surface primerhaving a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized second surface primers can be immobilizedto the support or immobilized to a coating on the support. Theimmobilized second surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized second surface primers are immobilized to a supportor immobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise a moietythat blocks primer extension (e.g., non-extendible terminal 3′ end),such as for example a phosphate group, a dideoxycytidine group, aninverted dT, or an amino group. The immobilized second surface primersare not extendible in a primer extension reaction. The immobilizedsecond surface primers lack a nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are joined or immobilized to an immobilizedfirst surface primer, and at least one portion of the individualconcatemer template molecule is hybridized to an immobilized secondsurface primer. The immobilized second surface primers serve to pin downa portion of the immobilized concatemer template molecules to thesupport (see FIGS. 12 and 24 ).

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (b): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (b) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers. The forward sequencing reactions cangenerate a plurality of extended forward sequencing primer strands. Insome embodiments, individual immobilized concatemer template moleculeshave multiple copies of the forward sequencing primer binding sites,wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site(e.g., see FIGS. 2 and 14 ). In some embodiments, the soluble forwardsequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble forward sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble forward sequencing primers lack a nucleotidehaving a scissile moiety. In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having a plurality ofnucleotide units attached to a core, where the multivalent molecules arelabeled with a detectable reporter moiety. In some embodiments, the coreis labeled with a detectable reporter moiety. In some embodiments, atleast one linker and/or at least one nucleotide unit of a nucleotide armis labeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (c): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized single stranded nucleic acidconcatemer template molecules. The plurality of extended forwardsequencing primer strands can be removed and replaced with a pluralityof forward extension strands by conducting a primer extension reaction(see FIGS. 3-5 , and FIGS. 15-17 ).

In some embodiments, step (c) comprises contacting at least one extendedforward sequencing primer strand with a plurality of strand displacingpolymerases and a plurality of nucleotides and in the absence of solubleamplification primers, under a condition suitable to conduct a stranddisplacing primer extension reaction using the at least one extendedforward sequencing primers strand to initiate the primer extensionreaction thereby generating a forward extension strand that iscovalently joined to the extended forward sequencing primers strand,wherein the forward extension strand is hybridized to the immobilizedconcatemer template molecule. For example, one of the extended forwardsequencing primer strands can serve as a primer for the stranddisplacing polymerase. The strand displacing polymerase can extend theextended forward sequencing primer strand, and displace downstreamextended forward sequencing primer strands while synthesizing anextended strand that replaces the downstream extended forward sequencingprimer strands (FIGS. 3 and 15 ). The newly extended strand iscovalently joined to an extended forward sequencing primer strand. Theimmobilized concatemer template molecules are retained.

The primer extension reaction can optionally include a plurality ofcompaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III)to generate forward extension strands. Individual forward extensionstrands can collapse into a nanoball having a more compact size and/orshape compared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (c) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble forward sequencing primers (e.g., a second plurality ofsoluble forward sequencing primers), a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble forward sequencing primers to the plurality of retainedimmobilized concatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the soluble sequencingprimers hybridize with the forward sequencing primer binding sequence inthe retained immobilized concatemer molecules (FIGS. 4 and 16 ). Theprimer extension reaction can optionally include a plurality ofcompaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III)to generate forward extension strands. Individual forward extensionstrands can collapse into a nanoball having a more compact size and/orshape compared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (c), the condition suitable to hybridizethe plurality of soluble forward sequencing primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, step (c) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble amplification primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the solubleamplification primers hybridize with the soluble amplification primerbinding sequence in the retained immobilized concatemer molecules (FIGS.5 and 17 ). The primer extension reaction can optionally include aplurality of compaction oligonucleotides and/or hexamine (e.g., cobalthexamine III) to generate forward extension strands. Individual forwardextension strands can collapse into a nanoball having a more compactsize and/or shape compared to a nanoball generated from a primerextension reaction conducted without compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III). Inclusion of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) in theprimer extension reaction can improve FWHM (full width half maximum) ofa spot image of the nanoball. The spot image can be represented as aGaussian spot and the size can be measured as a FWHM. A smaller spotsize as indicated by a smaller FWHM typically correlates with animproved image of the spot. In some embodiments, the FWHM of a nanoballspot can be about 10 μm or smaller.

In some embodiments, in step (c), the condition suitable to hybridizethe plurality of soluble amplification primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (c), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the primer extension polymerase of step (c)comprises a high fidelity polymerase. In some embodiments, the primerextension polymerase of step (c) comprises a DNA polymerase capable ofcatalyzing a primer extension reaction using a uracil-containingtemplate molecule (e.g., a uracil-tolerant polymerase). Exemplarypolymerases include, but are not limited to, Q5U Hot Start high-fidelityDNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNApolymerase, One Taq DNA polymerase (e.g., mixture of Taq and Deep VentDNA polymerases, catalog #M0480S from New England Biolabs), LongAmp TaqDNA polymerase (e.g., catalog #M0323S from New England Biolabs), EpimarkHot Start Taq DNA polymerase (e.g., catalog #M0490S from New EnglandBiolabs), Bst DNA polymerase (e.g., large fragment, catalog #M0275S fromNew England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog#M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog#M0269S from New England Biolabs), E. coli DNA polymerase (e.g., catalog#M0209S from New England Biolabs), Therminator DNA polymerase (e.g.,catalog #M0261S from New England Biolabs), Vent DNA polymerase and DeepVent DNA polymerase.

The pairwise methods described herein can provide increased accuracy ina downstream sequencing reaction because step (c) replaces the extendedforward sequencing primer strands that were generated in step (b) withforward extension strands having reduced base errors. The extendedforward sequencing primer strands are generated in step (b) and may ormay not contain erroneously incorporated nucleotides due topolymerase-catalyzed mis-paired bases. When step (c) is conducted with ahigh fidelity DNA polymerase, the resulting forward extension strandsmay have reduced base errors compared to the extended forward sequencingprimer strands. The forward extension strands will be used as a nucleicacid template for a downstream sequencing step (e.g., see step (e)below). Thus, step (c) can increase the sequencing accuracy of thedownstream step (e) and therefore increase the overall sequencingaccuracy of the pairwise sequencing workflow.

In some embodiments, the pairwise sequencing method further comprisesstep (d): removing the retained immobilized concatemer templatemolecules by generating abasic sites in the immobilized single strandedconcatemer template molecules at the nucleotide(s) having the scissilemoiety and generating gaps at the abasic sites to generate a pluralityof gap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized surface primers (FIGS. 6 and 18).

The abasic sites are generated on the retained concatemer templatestrands that contain nucleotides having scissile moieties. In someembodiments, the scissile moieties in the retained concatemer templatemolecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine. The abasic sites can be removed to generate a plurality ofsingle stranded nucleic acid template molecules having gaps whileretaining the plurality of forward extension strands. The abasic sitescan be generated by contacting the immobilized concatemer templatemolecules with an enzyme that removes the nucleo-base at the nucleotidehaving the scissile moiety. The uracil in the retained concatemertemplate strands can be converted to an abasic site using uracil DNAglycosylase (UDG). The 8oxoG in the retained concatemer template strandscan be converted to an abasic site using FPG glycosylase. Thedeoxyinosine in the retained concatemer template strands can beconverted to an abasic site using AlkA glycosylase.

In some embodiments, in step (d), the gaps can be generated bycontacting the abasic sites in the immobilized concatemer templatemolecules with an enzyme or a mixture of enzymes having lyase activitythat breaks the phosphodiester backbone at the 5′ and 3′ sides of theabasic site to release the base-free deoxyribose and generate a gap(FIGS. 6 and 18 ). The abasic sites can be removed using AP lyase, EndoIV endonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/APlyase. In some embodiments, generating the abasic sites and removal ofthe abasic sites to generate gaps can be achieved using a mixture ofuracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, forexample USER (Uracil-Specific Excision Reagent Enzyme from New EnglandBiolabs) or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (d), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical and/or heat.After the gap-removal procedure, the plurality of retained forwardextension strands (e.g., see FIGS. 7 and 9 , and FIGS. 19 and 21 ). ishybridized to the retained immobilized surface primers

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S). When a 5′ to 3′ double-stranded DNA exonuclease is used forremoving gap-containing template molecules, then the plurality ofsoluble amplification primers in step (c) can comprise at least onephosphorothioate diester bond at their 5′ ends which can render thesoluble amplification primers resistant to exonuclease degradation. Insome embodiments, the plurality of soluble amplification primers in step(c) comprise 2-5 or more consecutive phosphorothioate diester bonds attheir 5′ ends. In some embodiments, the plurality soluble amplificationprimers in step (c) comprise at least one ribonucleotide and/or at leastone 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotide which can renderthe forward sequencing primers resistant to exonuclease degradation.

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (e): sequencing the plurality of retained forward extension strandsthereby generating a plurality of extended reverse sequencing primerstrands. In some embodiments, the sequencing of step (e) comprisescontacting the plurality of retained forward extension strands with aplurality of soluble reverse sequencing primers under a conditionsuitable to hybridize the reverse sequencing primers to the reversesequencing primer binding site of the retained forward extensionstrands, and by conducting sequencing reactions using the hybridizedreverse sequencing primers wherein the forward sequencing reactionsgenerates a plurality of extended reverse sequencing primer strands(FIGS. 10 and 11 , and FIGS. 22 and 23 ). The extended reversesequencing primer strands are hybridized to the retained forwardextension strand. The retained forward extension strand is hybridized tothe first surface primer. The extended reverse sequencing primer strandsare not hybridized to the first surface primer, or covalently joined tothe first surface primer. Therefore, the extended reverse sequencingprimer strands are not immobilized to the support.

For the sake of simplicity, FIGS. 7 and 9 show exemplary retainedforward extension strands each having one copy of the sequence ofinterest and various universal primer binding sites. The skilled artisanwill appreciate that the retained forward extension strand can includetwo or more tandem copies containing the sequence of interest andvarious universal primer binding sites. Therefore, the reversesequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same retained forwardextension strand.

In some embodiments, in step (e), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)eth an e ford cacid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In an alternative embodiment, the sequencing of step (e) comprises usingthe immobilized surface primer as a sequencing primer and conductingsequencing reactions to generate a plurality of reverse sequencingstrands.

In some embodiments, the reverse sequencing reactions of step (e)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the retainedforward extension strands, one or more types of sequencing polymerases,and a plurality of nucleotides or a plurality of multivalent molecules.In some embodiments, the soluble reverse sequencing primers comprise 3′OH extendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules is described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site (e.g., seeFIGS. 10 and 11 , and FIGS. 22 and 23 ). In some embodiments, thesequencing reactions comprise a plurality of nucleotides (or analogsthereof) labeled with a detectable reporter moiety. In some embodiments,the sequencing reaction comprise a plurality of multivalent moleculeshaving nucleotide units, where the multivalent molecules are labeledwith a detectable reporter moiety. In some embodiments, the detectablereporter moiety comprises a fluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(e). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic add), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

On Support RCA and Pairwise Sequencing—Generating Abasic Sites

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a support having a plurality of surface primers(e.g., a plurality of first surface primers) immobilized thereon whereineach of the surface primers have a 3′ OH extendible end and lack anucleotide having a scissile moiety (FIG. 25 ). For example, the surfaceprimers lack uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) anddeoxyinosine. In some embodiments, the support comprises a plurality offirst surface primers. In some embodiments, the support lacks aplurality of second surface primers. In some embodiments, the supportcomprises a plurality of first and second surface primers.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The first surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of a nucleic acid library molecule (e.g., linear orcircular library molecules). The first surface primers can include aterminal 3′ nucleotide having a sugar 3′ OH moiety which is extendiblefor nucleotide polymerization (e.g., polymerase catalyzedpolymerization).

The immobilized first surface primers can be immobilized to the supportor immobilized to a coating on the support. The immobilized firstsurface primers can be embedded and attached (coupled) to the coating onthe support. In some embodiments, the 5′ end of the immobilized firstsurface primers are immobilized to a support or immobilized to a coatingon the support. Alternatively, an interior portion or the 3′ end of theimmobilized first surface primers can be immobilized to a support orimmobilized to a coating on the support. The support comprises aplurality of immobilized first surface primers having the same sequence.The immobilized first surface primers can be any length, for example4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, orlonger lengths.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the support further comprises a plurality of asecond surface primer immobilized thereon (FIG. 37 ). The second surfaceprimers have a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The second surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of an immobilized single stranded concatemer templatemolecule. The immobilized second surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedsecond surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized second surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized second surface primers comprise a 3′ non-extendible moiety.The 3′ terminal end of the immobilized second surface primers comprise amoiety that blocks primer extension, such as for example a phosphategroup, a dideoxycytidine group, an inverted dT, or an amino group. Theimmobilized second surface primers are not extendible in a primerextension reaction. The immobilized second surface primers lack anucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are covalently joined to an immobilizedfirst surface primer, and at least one portion of the individualconcatemer template molecule is hybridized to an immobilized secondsurface primer (FIG. 37 ). The immobilized second surface primers serveto pin down a portion of the immobilized concatemer template moleculesto the support. The immobilized concatemer template molecule has two ormore copies of a universal binding sequence for an immobilized secondsurface primer. The portion of the immobilized concatemer templatemolecule that includes the universal binding sequence for an immobilizedsecond surface primer can hybridize to the immobilized second surfaceprimer. In some embodiments, the second surface primers include aterminal 3′ blocking group that renders them non-extendible. In someembodiments, the second surface primers have terminal 3′ extendibleends.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (b): generating a plurality of immobilized single stranded nucleicacid concatemer template molecules wherein individual single strandednucleic acid concatemer template molecules are joined (e.g., covalentlyjoined) to an immobilized surface primer (e.g., an immobilized firstsurface primer), by hybridizing a plurality of single-stranded circularnucleic acid library molecules to the plurality of immobilized firstsurface primers and conducting a rolling circle amplification reactionwith a plurality of a strand displacing polymerase, and a plurality ofnucleotides which include dATP, dCTP, dGTP, dTTP and a nucleotide havinga scissile moiety, thereby generating a plurality of immobilized singlestranded nucleic acid concatemer template molecules (FIG. 26 ). In someembodiments, the rolling circle amplification reaction can be conductedin the presence, or in the absence, of a plurality of compactionoligonucleotides.

In some embodiments, the single-stranded circular nucleic acid librarymolecules comprise covalently closed circular molecules. In someembodiments, the single-stranded circular nucleic acid library moleculescan be removed from the concatemer template molecules with at least onewashing step which is conducted under a condition suitable to retain thesingle stranded nucleic acid concatemer template molecules whereindividual concatemer template molecules are operably joined to animmobilized first surface primer.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprise a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) a universalbinding sequence (or complementary sequence thereof) for a solubleforward sequencing primer, (ii) a universal binding sequence (orcomplementary sequence thereof) for a soluble reverse sequencing primer,(iii) a universal binding sequence (or complementary sequence thereof)for an immobilized first surface primer, (iv) a universal bindingsequence (or complementary sequence thereof) for an immobilized secondsurface primer, (v) a universal binding sequence (or complementarysequence thereof) for a first soluble amplification primer, (vi) auniversal binding sequence (or complementary sequence thereof) for asecond soluble amplification primer, (vii) a universal binding sequence(or complementary sequence thereof) for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the rolling circle amplification reaction of step(b) generates a plurality of immobilized single stranded nucleic acidconcatemer template molecules each comprising a concatemer having atleast one nucleotide having a scissile moiety and two or more copies ofa sequence of interest, and wherein the immobilized concatemer templatemolecules further comprise any one or any combination of two or more of:(i) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a soluble forward sequencing primer,(ii) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a soluble reverse sequencing primer,(iii) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for an immobilized first surface primer,(iv) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for an immobilized second surfaceprimer, (v) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a first soluble amplificationprimer, (vi) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a second soluble amplificationprimer, (vii) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a soluble compactionoligonucleotide, (viii) two or more copies of a sample barcode sequenceand/or (ix) two or more copies of a unique molecular index sequence.

In some embodiments, the plurality of immobilized single strandednucleic acid concatemer template molecules that are generated by therolling circle amplification reaction of step (b) further comprise twoor more copies of a universal binding sequence (or complementarysequence thereof) for immobilized second sequence surface primers. Insome embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are joined (e.g., covalently joined) to animmobilized first surface primer, and at least one portion of theindividual concatemer template molecule is hybridized to an immobilizedsecond surface primer. The immobilized second surface primers serve topin down a portion of the immobilized concatemer template molecules tothe support (see FIG. 37 ). In some embodiments, the second surfaceprimers include a terminal 3′ blocking group that renders themnon-extendible.

The rolling circle amplification reaction of step (b) can be conductedwith a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and anucleotide having a scissile moiety to generate immobilized concatemertemplate molecules which includes at least one nucleotide having ascissile moiety. The scissile moieties in the immobilized concatemertemplate molecules can be converted into abasic sites. In someembodiments, in the nucleotide mixture, the nucleotide having thescissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine (e.g.,8oxoG) or deoxyinosine. In the immobilized concatemer templatemolecules, the uridine can be converted to an abasic site using uracilDNA glycosylase (UDG), the 8oxoG can be converted to an abasic siteusing FPG glycosylase, and the deoxyinosine can be converted to anabasic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture can include an amount ofdUTP so that a target percent of the thymidine in the resultingconcatemer molecules are replaced with dUTP. For example, when 30% ofdTTP in the concatemer molecules are to be replaced with dUTP (e.g., 30%is the target percent) then the nucleotide mixture can contain 7.5% dUTP(e.g., 30/4=7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. Thetarget percent of dTTP to be replaced by dUTP can be about 0.1-1%, orabout 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about30-45%, or about 45-50%, or a higher percent of the dTTP in theimmobilized concatemer template molecules are replaced with nucleotideshaving a scissile moiety.

In some embodiments, the nucleotide mixture can include an amount ofdeoxyinosine so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with deoxyinosine. For example, when30% of dGTP in the concatemer molecules are to be replaced withdeoxyinosine (e.g., 30% is the target percent) then the nucleotidemixture can contain 7.5% deoxyinosine (e.g., 30/4=7.5%), 17.5% dGTP, and25% each for dATP, dCTP and dTTP. The target percent of dGTP to bereplaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or about5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about45-50%, or a higher percent of the dGTP in the immobilized concatemertemplate molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the nucleotide mixture can include an amount of8oxoG so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with 8oxoG. For example, when 30% ofdGTP in the concatemer molecules are to be replaced with 8oxoG (e.g.,30% is the target percent) then the nucleotide mixture can contain 7.5%8oxoG (e.g., 30/4=7.5%), 17.5% dGTP, and 25% each for dATP, dCTP anddTTP. The target percent of dGTP to be replaced by 8oxoG can be about0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%,or about 30-45%, or about 45-50%, or a higher percent of the dGTP in theimmobilized concatemer template molecules are replaced with nucleotideshaving a scissile moiety.

In some embodiments, the rolling circle amplification reaction generatesimmobilized concatemer template molecules with incorporated nucleotideshaving a scissile moiety that are distributed at random positions alongindividual immobilized concatemer template molecules. In someembodiments, the nucleotides having a scissile moiety are distributed atdifferent positions in the different immobilized concatemer templatemolecules.

In some embodiments, the pairwise sequencing method further comprisesstep (c): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (c) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers (FIG. 27 ). In some embodiments, the solubleforward sequencing primers comprise 3′ OH extendible ends. In someembodiments, the soluble forward sequencing primers comprise a 3′blocking moiety which can be removed to generate a 3′ OH extendible end.In some embodiments, the soluble forward sequencing primers lack anucleotide having a scissile moiety. The forward sequencing reactionscan generate a plurality of extended forward sequencing primer strands.In some embodiments, individual immobilized concatemer templatemolecules have multiple copies of the forward sequencing primer bindingsites, wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site(e.g., see FIG. 27 ). In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having a plurality ofnucleotide units attached to a core, where the multivalent molecules arelabeled with a detectable reporter moiety. In some embodiments, the coreis labeled with a detectable reporter moiety. In some embodiments, atleast one linker and/or at least one nucleotide unit of a nucleotide armis labeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (d): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized single stranded nucleic acidconcatemer template molecules. The plurality of extended forwardsequencing primer strands can be removed and replaced with a pluralityof forward extension strands by conducting a primer extension reaction(see FIGS. 28-30 ).

In some embodiments, step (d) comprises contacting at least one extendedforward sequencing primer strand with a plurality of strand displacingpolymerases and a plurality of nucleotides and in the absence of solubleamplification primers, under a condition suitable to conduct a stranddisplacing primer extension reaction using the at least one extendedforward sequencing primers strand to initiate the primer extensionreaction thereby generating a forward extension strand that iscovalently joined to the extended forward sequencing primers strand,wherein the forward extension strand is hybridized to the immobilizedconcatemer template molecule (FIG. 28 ). For example, one of theextended forward sequencing primer strands can serve as a primer for thestrand displacing polymerase. The strand displacing polymerase canextend the extended forward sequencing primer strand, and displacedownstream extended forward sequencing primer strands while synthesizingan extended strand that replaces the downstream extended forwardsequencing primer strands. The newly extended strand is covalentlyjoined to an extended forward sequencing primer strand. The immobilizedconcatemer template molecules are retained. The primer extensionreaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (d) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble forward sequencing primers (e.g., a second plurality ofsoluble forward sequencing primers), a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble forward sequencing primers to the plurality of retainedimmobilized concatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the soluble sequencingprimers hybridize with the forward sequencing primer binding sequence inthe retained immobilized concatemer molecules (FIG. 29 ). The primerextension reaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (d), the condition suitable to hybridizethe plurality of soluble forward sequencing primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, step (d) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble amplification primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the solubleamplification primers hybridize with the soluble amplification primerbinding sequence in the retained immobilized concatemer molecules (FIG.30 ). The primer extension reaction can optionally include a pluralityof compaction oligonucleotides and/or hexamine (e.g., cobalt hexamineIII) to generate forward extension strands. Individual forward extensionstrands can collapse into a nanoball having a more compact size and/orshape compared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (d), the condition suitable to hybridizethe plurality of soluble amplification primers to the plurality ofretained immobilized single standard nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (d), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or abuffering agent (e.g., Tris-HCl, MES, HEPES, or the like).

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the primer extension polymerase of step (d)comprises a high fidelity polymerase. In some embodiments, the primerextension polymerase of step (d) comprises a DNA polymerase capable ofcatalyzing a primer extension reaction using a uracil-containingtemplate molecule (e.g., a uracil-tolerant polymerase). Exemplarypolymerases include, but are not limited to, Q5U Hot Start high-fidelityDNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNApolymerase, One Taq DNA polymerase (e.g., mixture of Taq and Deep VentDNA polymerases, catalog #M0480S from New England Biolabs), LongAmp TaqDNA polymerase (e.g., catalog #M0323S from New England Biolabs), EpimarkHot Start Taq DNA polymerase (e.g., catalog #M0490S from New EnglandBiolabs), Bst DNA polymerase (e.g., large fragment, catalog #M0275S fromNew England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog#M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog#M0269S from New England Biolabs), E. coli DNA polymerase (e.g., catalog#M0209S from New England Biolabs), Therminator DNA polymerase (e.g.,catalog #M0261S from New England Biolabs), Vent DNA polymerase and DeepVent DNA polymerase.

The pairwise methods described herein can provide increased accuracy ina downstream sequencing reaction because step (d) replaces the extendedforward sequencing primer strands that were generated in step (c) withforward extension strands having reduced base errors. The extendedforward sequencing primer strands are generated in step (c) and may ormay not contain erroneously incorporated nucleotides due topolymerase-catalyzed mis-paired bases. When step (d) is conducted with ahigh fidelity DNA polymerase, the resulting forward extension strandsmay have reduced base errors compared to the extended forward sequencingprimer strands. The forward extension strands will be used as a nucleicacid template for a downstream sequencing step (e.g., see step (f)below). Thus, step (d) can increase the sequencing accuracy of thedownstream step (f) and therefore increase the overall sequencingaccuracy of the pairwise sequencing workflow.

In some embodiments, the pairwise sequencing method further comprisesstep (e): removing the retained immobilized concatemer templatemolecules by generating abasic sites in the immobilized single strandedconcatemer template molecules at the nucleotide(s) having the scissilemoiety and generating gaps at the abasic sites to generate a pluralityof gap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized surface primers (FIGS. 31 and 33).

The abasic sites are generated on the retained concatemer templatestrands that contain nucleotides having scissile moieties. In someembodiments, the scissile moieties in the retained concatemer templatemolecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine. The abasic sites can be removed to generate a plurality ofsingle stranded nucleic acid template molecules having gaps whileretaining the plurality of forward extension strands. The abasic sitescan be generated by contacting the immobilized concatemer templatemolecules with an enzyme that removes the nucleo-base at the nucleotidehaving the scissile moiety. The uracil in the retained concatemertemplate strands can be converted to an abasic site using uracil DNAglycosylase (UDG). The 8oxoG in the retained concatemer template strandscan be converted to an abasic site using FPG glycosylase. Thedeoxyinosine in the retained concatemer template strands can beconverted to an abasic site using AlkA glycosylase.

In some embodiments, in step (e), the gaps can be generated bycontacting the abasic sites in the immobilized concatemer templatemolecules with an enzyme or a mixture of enzymes having lyase activitythat breaks the phosphodiester backbone at the 5′ and 3′ sides of theabasic site to release the base-free deoxyribose and generate a gap(FIGS. 31 and 33 ). The abasic sites can be removed using AP lyase, EndoIV endonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/APlyase. In some embodiments, generating the abasic sites and removal ofthe abasic sites to generate gaps can be achieved using a mixture ofuracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, forexample USER (Uracil-Specific Excision Reagent Enzyme from New EnglandBiolabs) or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (e), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical compoundand/or heat. After the gap-removal procedure, the plurality of retainedforward extension strands are hybridized to the retained immobilizedsurface primers (FIGS. 32 and 34 ).

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S). When a 5′ to 3′ double-stranded DNA exonuclease is used forremoving gap-containing template molecules, then the plurality ofsoluble amplification primers in step (e) can comprise at least onephosphorothioate diester bond at their 5′ ends which can render thesoluble amplification primers resistant to exonuclease degradation. Insome embodiments, the plurality of soluble amplification primers in step(d) comprise 2-5 or more consecutive phosphorothioate diester bonds attheir 5′ ends. In some embodiments, the plurality soluble amplificationprimers in step (d) comprise at least one ribonucleotide and/or at leastone 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotide which can renderthe forward sequencing primers resistant to exonuclease degradation.

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (f): sequencing the plurality of retained forward extension strandsthereby generating a plurality of extended reverse sequencing primerstrands. In some embodiments, the sequencing of step (f) comprisescontacting the plurality of retained forward extension strands with aplurality of soluble reverse sequencing primers under a conditionsuitable to hybridize the reverse sequencing primers to the reversesequencing primer binding site of the retained forward extensionstrands, and by conducting sequencing reactions using the hybridizedreverse sequencing primers wherein the forward sequencing reactionsgenerates a plurality of extended reverse sequencing primer strands(FIGS. 35 and 36 ). The extended reverse sequencing primer strands arehybridized to the retained forward extension strand. The retainedforward extension strand is hybridized to the first surface primer. Theextended reverse sequencing primer strands are not hybridized to thefirst surface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support.

For the sake of simplicity, FIGS. 32 and 34 show exemplary retainedforward extension strands each having one copy of the sequence ofinterest and various universal primer binding sites. The skilled artisanwill appreciate that the retained forward extension strand can includetwo or more tandem copies containing the sequence of interest andvarious universal primer binding sites. Therefore, the reversesequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same retained forwardextension strand.

In some embodiments, in step (f), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In an alternative embodiment, the sequencing of step (f) comprises usingthe immobilized surface primer as a sequencing primer and conductingsequencing reactions to generate a plurality of reverse sequencingstrands.

In some embodiments, the reverse sequencing reactions of step (f)comprises contacting the plurality of reverse sequencing primers withthe reverse sequencing primer binding sequences of the retained forwardextension strands, one or more types of sequencing polymerases, and aplurality of nucleotides and/or a plurality of multivalent molecules. Insome embodiments, the soluble reverse sequencing primers comprise 3′ OHextendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules is described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site (e.g., seeFIGS. 35 and 36 ). In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having nucleotide units,where the multivalent molecules are labeled with a detectable reportermoiety. In some embodiments, the detectable reporter moiety comprises afluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(f). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA, (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2% 2or about 0.2-0.25%.

In Solution RCA and Pairwise Sequencing—Generating Abasic Sites

The present disclosure provides pairwise sequencing methods, comprisingstep (a): contacting in-solution a plurality of single-stranded circularnucleic acid library molecules to a plurality of soluble firstamplification primers, a plurality of a strand displacing polymerase,and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP anda nucleotide having a scissile moiety, under a condition suitable toform a plurality of library-primer duplexes and suitable for conductinga rolling circle amplification reaction, thereby generating a pluralityof single stranded nucleic acid concatemers having at least onenucleotide with a scissile moiety (FIG. 38 ). In some embodiments, thesoluble first amplification primer comprises a sequence that selectivelyhybridizes to a universal binding sequence in the circular nucleic acidlibrary molecules, such as for example a universal binding sequence (ora complementary sequence thereof) for the first soluble amplificationprimer. Alternatively, the soluble first amplification primer comprisesa random sequence that binds non-selectively to a sequence in thecircular nucleic acid library molecules.

In some embodiments, individual single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence (or acomplementary sequence thereof) for a soluble forward sequencing primer,(ii) a universal binding sequence (or a complementary sequence thereof)for a soluble reverse sequencing primer, (iii) a universal bindingsequence (or a complementary sequence thereof) for an immobilized firstsurface primer, (iv) a universal binding sequence (or a complementarysequence thereof) for an immobilized second surface primer, (v) auniversal binding sequence (or a complementary sequence thereof) for afirst soluble amplification primer, (vi) a universal binding sequence(or a complementary sequence thereof) for a second soluble amplificationprimer, (vii) a universal binding sequence (or a complementary sequencethereof) for a soluble compaction oligonucleotide, (viii) a samplebarcode sequence and/or (ix) a unique molecular index sequence. In someembodiments, the single-stranded circular nucleic acid library moleculescomprise covalently closed circular molecules.

In some embodiments, the rolling circle amplification reaction of step(a) generates a plurality of single stranded nucleic acid concatemermolecules in solution, comprising a concatemer having at least onenucleotide having a scissile moiety. In some embodiments, individualconcatemer template molecules in the plurality comprise two or morecopies of a sequence of interest, and wherein the individual immobilizedconcatemer template molecules further comprise any one or anycombination of two or more of: (i) two or more copies of a universalbinding sequence for a soluble forward sequencing primer, (ii) two ormore copies of a universal binding sequence for a soluble reversesequencing primer, (iii) two or more copies of a universal bindingsequence for an immobilized first surface primer, (iv) two or morecopies of a universal binding sequence for an immobilized second surfaceprimer, (v) two or more copies of a universal binding sequence for afirst soluble amplification primer, (vi) two or more copies of auniversal binding sequence for a second soluble amplification primer,(vii) two or more copies of a universal binding sequence for a solublecompaction oligonucleotide, (viii) two or more copies of a samplebarcode sequence and/or (ix) two or more copies of a unique molecularindex sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

The in-solution rolling circle amplification reaction of step (a) can beconducted with a nucleotide mixture containing dATP, dCTP, dGTP, dTTPand a nucleotide having a scissile moiety to generate the concatemermolecules which includes at least one nucleotide having a scissilemoiety. The scissile moieties in the concatemer molecules can beconverted into abasic sites. In some embodiments, in the nucleotidemixture, the nucleotide having the scissile moiety comprises uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. In theconcatemer molecules, the uridine can be converted to an abasic siteusing uracil DNA glycosylase (UDG), the 8oxoG can be converted to anabasic site using FPG glycosylase, and the deoxyinosine can be convertedto an abasic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture can include an amount ofdUTP so that a target percent of the thymidine in the resultingconcatemer molecules are replaced with dUTP. For example, when 30% ofdTTP in the concatemer molecules are to be replaced with dUTP (e.g., 30%is the target percent) then the nucleotide mixture can contain 7.5% dUTP(e.g., 30/4=7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. Thetarget percent of dTTP to be replaced by dUTP can be about 0.1-1%, orabout 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about30-45%, or about 45-50%, or a higher percent of the dTTP in theconcatemer molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the nucleotide mixture can include an amount ofdeoxyinosine so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with deoxyinosine. For example, when30% of dGTP in the concatemer molecules are to be replaced withdeoxyinosine (e.g., 30% is the target percent) then the nucleotidemixture can contain 7.5% deoxyinosine (e.g., 30/4=7.5%), 17.5% dGTP, and25% each for dATP, dCTP and dTTP. The target percent of dGTP to bereplaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or about5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about45-50%, or a higher percent of the dGTP in the concatemer molecules arereplaced with nucleotides having a scissile moiety.

In some embodiments, the nucleotide mixture can include an amount of8oxoG so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with 8oxoG. For example, when 30% ofdGTP in the concatemer molecules are to be replaced with 8oxoG (e.g.,30% is the target percent) then the nucleotide mixture can contain 7.5%8oxoG (e.g., 30/4=7.5%), 17.5% dGTP, and 25% each for dATP, dCTP anddTTP. The target percent of dGTP to be replaced by 8oxoG can be about0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%,or about 30-45%, or about 45-50%, or a higher percent of the dGTP in theconcatemer molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the in-solution rolling circle amplificationreaction generates concatemer molecules with incorporated nucleotideshaving a scissile moiety that are distributed at random positions alongindividual immobilized concatemer template molecules. In someembodiments, the nucleotides having a scissile moiety are distributed atdifferent positions in the different concatemer molecules.

In some embodiments, the pairwise sequencing method further comprisesstep (b): distributing the rolling circle amplification reaction fromstep (a) onto a support having a plurality of the first surface primersimmobilized thereon, under a condition suitable for hybridizing one ormore portions of individual single stranded concatemers to one or moreimmobilized first surface primers (FIG. 39 ). In some embodiments, theimmobilized first surface primers have terminal 3′ group that arenon-extendible. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a moiety that blocks primerextension, such as for example a phosphate group, a dideoxycytidinegroup, an inverted dT, or an amino group. In some embodiments, theimmobilized first surface primer have an extendible 3′OH end. In someembodiments, the immobilized first surface primers lack a nucleotidehaving a scissile moiety. The concatemers are immobilized to the supportby hybridization to the immobilized first surface primers. In someembodiments, the support comprises a plurality of first surface primers.In some embodiments, the support lacks a plurality of second surfaceprimers. In some embodiments, the support comprises a plurality of firstand second surface primers.

In some embodiments, the pairwise sequencing method further comprisesstep (c): continuing the rolling circle amplification reaction on thesupport to generate a plurality of extended concatemer templatemolecules that are immobilized via hybridization to the immobilizedfirst surface primers (FIG. 40 ). The on-support RCA reaction can beconducted with a plurality of a strand displacing polymerase, and aplurality of nucleotides which include dATP, dCTP, dGTP, dTTP and anucleotide having a scissile moiety, under a condition suitable togenerate a plurality of extended concatemers having at least onenucleotide with a scissile moiety (FIG. 41 ). In some embodiments, therolling circle amplification reaction on the support can be conducted inthe presence, or in the absence, of a plurality of compactionoligonucleotides.

In some embodiments, the on-support rolling circle amplificationreaction generates immobilized concatemer template molecules withincorporated nucleotides having a scissile moiety that are distributedat random positions along individual immobilized concatemer templatemolecules. In some embodiments, the nucleotides having a scissile moietyare distributed at different positions in the different immobilizedconcatemer template molecules.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The first surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of the concatemer molecules. In some embodiments, thefirst surface primers can lack a terminal 3′ OH extendible end whichrenders the first surface primers non-extendible. In some embodiments,the first surface primers include a terminal 3′ OH group which isextendible for nucleotide polymerization (e.g., polymerase catalyzedpolymerization). The immobilized first surface primers can beimmobilized to the support or immobilized to a coating on the support.The immobilized first surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the plurality of immobilized first surface primerscomprise 3′ extendible ends. In some embodiments, the 3′ terminal end ofthe immobilized first surface primers comprise a moiety that blocksprimer extension, such as for example a phosphate group, adideoxycytidine group, an inverted dT, or an amino group. In someembodiments, the immobilized first surface primers are not extendible ina primer extension reaction. The immobilized first surface primers lacka nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the concatemer template molecules.

In some embodiments, the support further comprises a plurality of asecond surface primer immobilized thereon (FIG. 52 ). The second surfaceprimers have a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers comprise single strandedoligonucleotides comprising DNA, RNA or a combination of DNA and RNA.The second surface primers comprise a sequence that is whollycomplementary or partially complementary along their lengths to at leasta portion of a concatemer molecule. The immobilized second surfaceprimers can be immobilized to the support or immobilized to a coating onthe support. The immobilized second surface primers can be embedded andattached (coupled) to the coating on the support. In some embodiments,the 5′ end of the immobilized second surface primers are immobilized toa support or immobilized to a coating on the support. Alternatively, aninterior portion or the 3′ end of the immobilized second surface primerscan be immobilized to a support or immobilized to a coating on thesupport. The support comprises a plurality of immobilized second surfaceprimers having the same sequence. The immobilized second surface primerscan be any length, for example 4-50 nucleotides, or 50-100 nucleotides,or 100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. The 3′ terminal end of theimmobilized second surface primers comprise a moiety that blocks primerextension, such as for example a phosphate group, a dideoxycytidinegroup, an inverted dT, or an amino group. The immobilized second surfaceprimers are not extendible in a primer extension reaction. Theimmobilized second surface primers lack a nucleotide having a scissilemoiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are hybridized to an immobilized firstsurface primer, and at least one portion of the individual concatemertemplate molecule is hybridized to an immobilized second surface primer(FIG. 52 ). The immobilized second surface primers serve to pin down aportion of the immobilized concatemer template molecules to the support.The immobilized concatemer template molecule has two or more copies of auniversal binding sequence for an immobilized second surface primer. Theportion of the immobilized concatemer template molecule that includesthe universal binding sequence for an immobilized second surface primercan hybridize to the immobilized second surface primer. In someembodiments, the second surface primers include a terminal 3′ blockinggroup that renders them non-extendible. In some embodiments, the secondsurface primers have terminal 3′ extendible ends.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers react with the solutions in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (d): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (d) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers. In some embodiments, the soluble forwardsequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble forward sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble forward sequencing primers lack a nucleotidehaving a scissile moiety. The forward sequencing reactions can generatea plurality of extended forward sequencing primer strands (FIG. 42 ). Insome embodiments, individual immobilized concatemer template moleculeshave multiple copies of the forward sequencing primer binding sites,wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site(e.g., see FIG. 42 ). In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having a plurality ofnucleotide units attached to a core, where the multivalent molecules arelabeled with a detectable reporter moiety. In some embodiments, the coreis labeled with a detectable reporter moiety. In some embodiments, atleast one linker and/or at least one nucleotide unit of a nucleotide armis labeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (e): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized single stranded nucleic acidconcatemer template molecules. The plurality of extended forwardsequencing primer strands can be removed and replaced with a pluralityof forward extension strands by conducting a primer extension reaction(See FIGS. 43-45 ).

In some embodiments, step (e) comprises contacting at least one extendedforward sequencing primer strand with a plurality of strand displacingpolymerases and a plurality of nucleotides and in the absence of solubleamplification primers, under a condition suitable to conduct a stranddisplacing primer extension reaction using the at least one extendedforward sequencing primers strand to initiate the primer extensionreaction thereby generating a forward extension strand that iscovalently joined to the extended forward sequencing primers strand,wherein the forward extension strand is hybridized to the immobilizedconcatemer template molecule (FIG. 43 ). For example, one of theextended forward sequencing primer strands can serve as a primer for thestrand displacing polymerase. The strand displacing polymerase canextend the extended forward sequencing primer strand, and displacedownstream extended forward sequencing primer strands while synthesizingan extended strand that replaces the downstream extended forwardsequencing primer strands. The newly extended strand is covalentlyjoined to an extended forward sequencing primer strand. The immobilizedconcatemer template molecules are retained. The primer extensionreaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (e) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble forward sequencing primers (e.g., a second plurality ofsoluble forward sequencing primers), a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble forward sequencing primers to the plurality of retainedimmobilized concatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the soluble sequencingprimers hybridize with the forward sequencing primer binding sequence inthe retained immobilized concatemer molecules (FIG. 44 ). The primerextension reaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (e), the condition suitable to hybridizethe plurality of soluble forward sequencing primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, step (e) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble amplification primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the solubleamplification primers hybridize with the soluble amplification primerbinding sequence in the retained immobilized concatemer molecules (FIG.45 ). The primer extension reaction can optionally include a pluralityof compaction oligonucleotides and/or hexamine (e.g., cobalt hexamineIII) to generate forward extension strands. Individual forward extensionstrands can collapse into a nanoball having a more compact size and/orshape compared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (e), the condition suitable to hybridizethe plurality of soluable amplification primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (e), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or abuffering agent (e.g., Tris-HCl, MES, HEPES, or the like).

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the primer extension polymerase of step (e)comprises a high fidelity polymerase. In some embodiments, the primerextension polymerase of step (e) comprises a DNA polymerase capable ofcatalyzing a primer extension reaction using a uracil-containingtemplate molecule (e.g., a uracil-tolerant polymerase). Exemplarypolymerases include, but are not limited to, Q5U Hot Start high-fidelityDNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNApolymerase, One Taq DNA polymerase (e.g., mixture of Taq and Deep VentDNA polymerases, catalog #M0480S from New England Biolabs), LongAmp TaqDNA polymerase (e.g., catalog #M0323S from New England Biolabs), EpimarkHot Start Taq DNA polymerase (e.g., catalog #M0490S from New EnglandBiolabs), Bst DNA polymerase (e.g., large fragment, catalog #M0275S fromNew England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog#M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog#M0269S from New England Biolabs), E. coli DNA polymerase (e.g., catalog#M0209S from New England Biolabs), Therminator DNA polymerase (e.g.,catalog #M0261S from New England Biolabs), Vent DNA polymerase and DeepVent DNA polymerase.

The pairwise methods described herein can provide increased accuracy ina downstream sequencing reaction because step (e) replaces the extendedforward sequencing primer strands that were generated in step (d) withforward extension strands having reduced base errors. The extendedforward sequencing primer strands are generated in step (d) and may ormay not contain erroneously incorporated nucleotides due topolymerase-catalyzed mis-paired bases. When step (e) is conducted with ahigh fidelity DNA polymerase, the resulting forward extension strandsmay have reduced base errors compared to the extended forward sequencingprimer strands. The forward extension strands will be used as a nucleicacid template for a downstream sequencing step (e.g., see step (f)below). Thus, step (e) can increase the sequencing accuracy of thedownstream step (g) and therefore increase the overall sequencingaccuracy of the pairwise sequencing workflow.

In some embodiments, the pairwise sequencing method further comprisesstep (f): removing the retained immobilized concatemer templatemolecules by generating abasic sites in the immobilized single strandedconcatemer template molecules at the nucleotide(s) having the scissilemoiety and generating gaps at the abasic sites to generate a pluralityof gap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized surface primers (FIGS. 46 and 48).

The abasic sites are generated on the retained concatemer templatestrands that contain nucleotides having scissile moieties. In someembodiments, the scissile moieties in the retained concatemer templatemolecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine. The abasic sites can be removed to generate a plurality ofsingle stranded nucleic acid template molecules having gaps whileretaining the plurality of forward extension strands. The abasic sitescan be generated by contacting the immobilized concatemer templatemolecules with an enzyme that removes the nucleo-base at the nucleotidehaving the scissile moiety. The uracil in the retained concatemertemplate strands can be converted to an abasic site using uracil DNAglycosylase (UDG). The 8oxoG in the retained concatemer template strandscan be converted to an abasic site using FPG glycosylase. Thedeoxyinosine in the retained concatemer template strands can beconverted to an abasic site using AlkA glycosylase.

In some embodiments, in step (f), the gaps can be generated bycontacting the abasic sites in the immobilized concatemer templatemolecules with an enzyme or a mixture of enzymes having lyase activitythat breaks the phosphodiester backbone at the 5′ and 3′ sides of theabasic site to release the base-free deoxyribose and generate a gap(FIGS. 46 and 48 ). The abasic sites can be removed using AP lyase, EndoIV endonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/APlyase. In some embodiments, generating the abasic sites and removal ofthe abasic sites to generate gaps can be achieved using a mixture ofuracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, forexample USER (Uracil-Specific Excision Reagent Enzyme from New EnglandBiolabs) or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (f), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical compoundand/or heat. After the gap-removal procedure, the plurality of retainedforward extension strands can be hybridized to the retained immobilizedsurface primers (FIGS. 47 and 49 ).

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S). When a 5′ to 3′ double-stranded DNA exonuclease is used forremoving gap-containing template molecules, then the plurality ofsoluble amplification primers in step (e) can comprise at least onephosphorothioate diester bond at their 5′ ends which can render thesoluble amplification primers resistant to exonuclease degradation. Insome embodiments, the plurality of soluble amplification primers in step(e) comprise 2-5 or more consecutive phosphorothioate diester bonds attheir 5′ ends. In some embodiments, the plurality soluble amplificationprimers in step (e) comprise at least one ribonucleotide and/or at leastone 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotide which can renderthe forward sequencing primers resistant to exonuclease degradation.

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (g): sequencing the plurality of retained forward extension strandsthereby generating a plurality of extended reverse sequencing primerstrands. In some embodiments, the sequencing of step (g) comprisescontacting the plurality of retained forward extension strands with aplurality of soluble reverse sequencing primers under a conditionsuitable to hybridize the reverse sequencing primers to the reversesequencing primer binding site of the retained forward extensionstrands, and by conducting sequencing reactions using the hybridizedreverse sequencing primers wherein the forward sequencing reactionsgenerates a plurality of extended reverse sequencing primer strands(FIGS. 50 and 51 ). The extended reverse sequencing primer strands arehybridized to the retained forward extension strand. The retainedforward extension strand is hybridized to the first surface primer. Theextended reverse sequencing primer strands are not hybridized to thefirst surface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support.

For the sake of simplicity, FIGS. 47 and 49 show exemplary retainedforward extension strands each having either (i) one copy of thesequence of interest and various universal primer binding sites (FIG. 47) or (ii) two tandem copies of the sequence of interest and variousuniversal primer binding sites (FIG. 49 ). The skilled artisan willappreciate that the retained forward extension strand can include two,three, four or many more tandem copies containing the sequence ofinterest and various universal primer binding sites. Therefore, thereverse sequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same retained forwardextension strand.

In some embodiments, in step (g), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In an alternative embodiment, the sequencing of step (g) comprises usingthe immobilized surface primer as a sequencing primer and conductingsequencing reactions to generate a plurality of reverse sequencingstrands.

In some embodiments, the reverse sequencing reactions of step (g)comprises contacting the plurality of reverse sequencing primers withthe reverse sequencing primer binding sequences of the retained forwardextension strands, one or more types of sequencing polymerases, and aplurality of nucleotides and/or a plurality of multivalent molecules. Insome embodiments, the soluble reverse sequencing primers comprise 3′ OHextendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules is described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site (FIGS. 50 and51 ). In some embodiments, the sequencing reactions comprise a pluralityof nucleotides (or analogs thereof) labeled with a detectable reportermoiety. In some embodiments, the sequencing reaction comprise aplurality of multivalent molecules having nucleotide units, where themultivalent molecules are labeled with a detectable reporter moiety. Insome embodiments, the detectable reporter moiety comprises afluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(g). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA, (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetraacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

On Support Ligation and RCA and Pairwise Sequencing

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a support having a plurality of surface primers(e.g., a plurality of first surface primers) immobilized thereon,wherein individual first surface primers in the plurality comprise afirst portion (SP1-A) and a second portion (SP1-B), and the individualfirst surface primers comprising a 3′ extendible end and lacking anucleotide having a scissile moiety that can be cleaved to generate anabasic site in the first surface primer. In some embodiments, theimmobilized first surface primers lack a nucleotide having a scissilemoiety (FIG. 55 ). For example, the surface primers lack uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine. In someembodiments, the first and second portions (SP1-A and SP1-B) of thefirst surface primers have the same or different lengths. The firstportion (SP1-A) of the first surface primers can be about 4-50nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, or longerlengths. The second portion (SP1-B) of the first surface primers can beabout 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides,or longer lengths. In some embodiments, the first and second portions(SP1-A and SP1-B) of the immobilized first surface primers have the sameor different sequences. In some embodiments, the support comprises aplurality of first surface primers. In some embodiments, the supportlacks a plurality of second surface primers. In some embodiments, thesupport comprises a plurality of first and second surface primers.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The first surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of a nucleic acid library molecule (e.g., linear orcircular library molecules). The first surface primers can include aterminal 3′ nucleotide having a sugar 3′ OH moiety which is extendiblefor nucleotide polymerization (e.g., polymerase catalyzedpolymerization).

The immobilized first surface primers can be immobilized to the supportor immobilized to a coating on the support. The immobilized firstsurface primers can be embedded and attached (coupled) to the coating onthe support. In some embodiments, the 5′ end of the immobilized firstsurface primers are immobilized to a support or immobilized to a coatingon the support. Alternatively, an interior portion or the 3′ end of theimmobilized first surface primers can be immobilized to a support orimmobilized to a coating on the support. The support comprises aplurality of immobilized first surface primers having the same sequence.The immobilized first surface primers can be any length, for example4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, orlonger lengths.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the support further comprises a plurality of asecond surface primer immobilized thereon (FIG. 72 ). The second surfaceprimers have a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The second surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of an immobilized single stranded concatemer templatemolecule. The immobilized second surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedsecond surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized second surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized second surface primers comprise a 3′ non-extendible moiety.The 3′ terminal end of the immobilized second surface primers comprise amoiety that blocks primer extension, such as for example a phosphategroup, a dideoxycytidine group, an inverted dT, or an amino group. Theimmobilized second surface primers are not extendible in a primerextension reaction. The immobilized second surface primers lack anucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are covalently joined to an immobilizedfirst surface primer, and at least one portion of the individualconcatemer template molecule is hybridized to an immobilized secondsurface primer (FIG. 72 ). The immobilized second surface primers serveto pin down a portion of the immobilized concatemer template moleculesto the support. The immobilized concatemer template molecule has two ormore copies of a universal binding sequence for an immobilized secondsurface primer. The portion of the immobilized concatemer templatemolecule that includes the universal binding sequence for an immobilizedsecond surface primer can hybridize to the immobilized second surfaceprimer. In some embodiments, the second surface primers include aterminal 3′ blocking group that renders them non-extendible. In someembodiments, the second surface primers have terminal 3′ extendibleends.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (b): contacting the plurality of the first surface primers with aplurality of single stranded linear nucleic acid library molecules eachlibrary molecule having 5′ and 3′ ends. The contacting is conductedunder a condition suitable for hybridizing individual library moleculesto an immobilized first surface primer to form a circularized librarymolecule having a gap or nick between the 5′ and 3′ ends of thecircularized library molecule (FIGS. 57 and 58 ).

In some embodiments, the position of the gap or nick in the circularizedlibrary molecules can be asymmetrical or symmetrical relative to theduplex formed by hybridizing the 5′ and 3′ ends of the linear librarymolecule to the immobilized first surface primers. For example, FIG. 57shows an asymmetrical positioned gap or nick. FIG. 58 (left) shows anasymmetrical positioned gap or nick. FIG. 58 (right) shows a symmetricalpositioned gap or nick. An asymmetrical or symmetrical positionedgap/nick can be generated by adjusting the length of the first portion(SP1-A) and the second portion (SP1-B) in the immobilized first surfaceprimers.

In some embodiments, individual library molecules in the pluralitycomprise a sequence of interest and the library molecules furthercomprise any one or any combination of two or more of: (i) a universalbinding sequence (or complementary sequence thereof) for a solubleforward sequencing primer; (ii) a universal binding sequence (orcomplementary sequence thereof) for a soluble reverse sequencing primer;(iii) a universal binding sequence (or complementary sequence thereof)for a first portion of an immobilized first surface primer (SP1-A); (iv)a universal binding sequence (or complementary sequence thereof) for asecond portion of an immobilized first surface primer (SP1-B); (v) auniversal binding sequence (or complementary sequence thereof) for animmobilized second surface primer; (vi) a universal binding sequence (orcomplementary sequence thereof) for a first soluble amplificationprimer; (vii) a universal binding sequence (or complementary sequencethereof) for a second soluble amplification primer; (viii) a universalbinding sequence (or complementary sequence thereof) for a solublecompaction oligonucleotide; (ix) a sample barcode sequence and/or (x) aunique molecular index sequence. An exemplary single stranded linearlibrary molecule is shown in FIG. 56 .

In some embodiments, the universal binding sequence for a first portionof an immobilized first surface primer (e.g., SP1-A′) in the linearlibrary molecule can hybridize to the first portion of the immobilizedfirst surface primer (SP1-A). In some embodiments, the universal bindingsequence for a second portion of an immobilized first surface primer(e.g., SP1-B′) in the linear library molecule can hybridize to thesecond portion of the immobilized first surface primer (SP1-B). In someembodiments, the immobilized first surface primers comprise a firstportion (SP1-A) and a second portion (SP1-B) which hybridize to SP1-A′and SP1-B′ in the linear library molecule, and the first surface primersserve as a nucleic acid splint molecule for circularizing the linearlibrary molecules.

In some embodiments, the pairwise sequencing method further comprisesstep (c): enzymatically closing the gap or nick thereby formingindividual single stranded covalently closed circular molecules that arehybridized to an immobilized first surface primer (FIG. 59 , FIG. 60(left) and FIG. 60 (right)).

In some embodiments, the gap in the circularized library molecule isclosed by conducting a polymerase-catalyzed gap fill-in reaction usingthe 3′ extendible end of the library molecule as an initiation site forthe polymerase-catalyzed fill-in reaction and using the immobilizedfirst surface primer as a template molecule thereby forming circularizedmolecule having a nick. The nick is closed by conducting an enzymaticligation reaction to form a single stranded covalently closed circularmolecule, wherein individual covalently closed circular molecules arehybridized to an immobilized first surface primer. In some embodiments,the gap fill-in reaction can be conducting with a plurality ofnucleotides and a polymerase that lacks 5′ to 3′ strand displacementactivity. The polymerase comprises E. coli DNA polymerase I, Klenowfragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4 DNApolymerase. In some embodiments, the ligation reaction can be conductedusing a DNA ligase which comprises a T3, T4, T7 or Taq DNA ligase.

In some embodiments, the nick in the circularized library molecule isclosed by conducting a ligase-catalyzed ligation reaction to form asingle stranded covalently closed circular molecule, wherein individualcovalently closed circular molecules are hybridized to an immobilizedfirst surface primer. In some embodiments, the ligase enzyme comprisesT3, T4, T7 or Taq DNA ligase.

In some embodiments, the pairwise sequencing method further comprisesstep (d): generating a plurality of immobilized single stranded nucleicacid concatemer template molecules by conducting a rolling circleamplification reaction with a plurality of a strand displacingpolymerase, and a plurality of nucleotides which include dATP, dCTP,dGTP, dTTP and a nucleotide having a scissile moiety that can be cleavedto generate an abasic site, thereby generating a plurality ofimmobilized single stranded nucleic acid concatemer template moleculeshaving at least one nucleotide with a scissile moiety, whereinindividual single stranded nucleic acid concatemer template moleculesare covalently joined to an immobilized first surface primer (FIG. 61 ).In some embodiments, the rolling circle amplification reaction can beconducted in the presence, or in the absence, of a plurality of aplurality of compaction oligonucleotides.

In some embodiments, the single-stranded circular nucleic acid librarymolecules can be removed from the concatemer template molecules with atleast one washing step which is conducted under a condition suitable toretain the single stranded nucleic acid concatemer template moleculeswhere individual concatemer template molecules are operably joined to animmobilized first surface primer.

In some embodiments, individual immobilized concatemer templatemolecules generated by the rolling circle amplification reactioncomprise two or more copies of a sequence of interest and wherein theindividual immobilized concatemer template molecules further compriseany one or any combination of two or more of: (i) two or more copies ofa universal binding sequence for a soluble forward sequencing primer;(ii) two or more copies of a universal binding sequence for a solublereverse sequencing primer; (iii) two or more copies of a universalbinding sequence for a first portion of an immobilized first surfaceprimer (SP1-A); (iv) two or more copies of a universal binding sequencefor a second portion of an immobilized first surface primer (SP1-B); (v)two or more copies of a universal binding sequence for an immobilizedsecond surface primer; (vi) two or more copies of a universal bindingsequence for a first soluble amplification primer (vii) two or morecopies of a universal binding sequence for a second solubleamplification primer; (viii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide; (ix) two or morecopies of a sample barcode sequence and/or (x) two or more copies of aunique molecular index sequence.

In some embodiments, the plurality of immobilized single strandednucleic acid concatemer template molecules that are generated by therolling circle amplification reaction of step (d) further comprise twoor more copies of a universal binding sequence (or complementarysequence thereof) for immobilized second sequence surface primers. Insome embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are joined (e.g., covalently joined) to animmobilized first surface primer, and at least one portion of theindividual concatemer template molecule is hybridized to an immobilizedsecond surface primer. The immobilized second surface primers serve topin down a portion of the immobilized concatemer template molecules tothe support (see FIG. 72 ). In some embodiments, the second surfaceprimers include a terminal 3′ blocking group that renders themnon-extendible.

The rolling circle amplification reaction of step (d) can be conductedwith a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and anucleotide having a scissile moiety to generate immobilized concatemertemplate molecules which includes at least one nucleotide having ascissile moiety. The scissile moieties in the immobilized concatemertemplate molecules can be converted into abasic sites. In someembodiments, in the nucleotide mixture, the nucleotide having thescissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine (e.g.,8oxoG) or deoxyinosine. In the immobilized concatemer templatemolecules, the uridine can be converted to an abasic site using uracilDNA glycosylase (UDG), the 8oxoG can be converted to an abasic siteusing FPG glycosylase, and the deoxyinosine can be converted to anabasic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture can include an amount ofdUTP so that a target percent of the thymidine in the resultingconcatemer molecules are replaced with dUTP. For example, when 30% ofdTTP in the concatemer molecules are to be replaced with dUTP (e.g., 30%is the target percent) then the nucleotide mixture can contain 7.5% dUTP(e.g., 30/4=7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. Thetarget percent of dTTP to be replaced by dUTP can be about 0.1-1%, orabout 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about30-45%, or about 45-50%, or a higher percent of the dTTP in theimmobilized concatemer template molecules are replaced with nucleotideshaving a scissile moiety.

In some embodiments, the nucleotide mixture can include an amount ofdeoxyinosine so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with deoxyinosine. For example, when30% of dGTP in the concatemer molecules are to be replaced withdeoxyinosine (e.g., 30% is the target percent) then the nucleotidemixture can contain 7.5% deoxyinosine (e.g., 30/4=7.5%), 17.5% dGTP, and25% each for dATP, dCTP and dTTP. The target percent of dGTP to bereplaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or about5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about45-50%, or a higher percent of the dGTP in the immobilized concatemertemplate molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the nucleotide mixture can include an amount of8oxoG so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with 8oxoG. For example, when 30% ofdGTP in the concatemer molecules are to be replaced with 8oxoG (e.g.,30% is the target percent) then the nucleotide mixture can contain 7.5%8oxoG (e.g., 30/4=7.5%), 17.5% dGTP, and 25% each for dATP, dCTP anddTTP. The target percent of dGTP to be replaced by 8oxoG can be about0.1-1%, or about 1-5%, or about 5-10%, or about or about 20-30%, orabout 30-45%, or about 45-50%, or a higher percent of the dGTP in theimmobilized concatemer template molecules are replaced with nucleotideshaving a scissile moiety.

In some embodiments, the rolling circle amplification reaction generatesimmobilized concatemer template molecules with incorporated nucleotideshaving a scissile moiety that are distributed at random positions alongindividual immobilized concatemer template molecules. In someembodiments, the nucleotides having a scissile moiety are distributed atdifferent positions in the different immobilized concatemer templatemolecules.

In some embodiments, the pairwise sequencing method further comprisesstep (e): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (e) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers (FIG. 62 ). In some embodiments, the solubleforward sequencing primers comprise 3′ OH extendible ends. In someembodiments, the soluble forward sequencing primers comprise a 3′blocking moiety which can be removed to generate a 3′ OH extendible end.In some embodiments, the soluble forward sequencing primers lack anucleotide having a scissile moiety. The forward sequencing reactionscan generate a plurality of extended forward sequencing primer strands.In some embodiments, individual immobilized concatemer templatemolecules have multiple copies of the forward sequencing primer bindingsites, wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site(e.g., see FIG. 62 ). In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having a plurality ofnucleotide units attached to a core, where the multivalent molecules arelabeled with a detectable reporter moiety. In some embodiments, the coreis labeled with a detectable reporter moiety. In some embodiments, atleast one linker and/or at least one nucleotide unit of a nucleotide armis labeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (f): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized single stranded nucleic acidconcatemer template molecules. The plurality of extended forwardsequencing primer strands can be removed and replaced with a pluralityof forward extension strands by conducting a primer extension reaction(see FIGS. 63-65 ).

In some embodiments, step (f) comprises contacting at least one extendedforward sequencing primer strand with a plurality of strand displacingpolymerases and a plurality of nucleotides and in the absence of solubleamplification primers, under a condition suitable to conduct a stranddisplacing primer extension reaction using the at least one extendedforward sequencing primers strand to initiate the primer extensionreaction thereby generating a forward extension strand that iscovalently joined to the extended forward sequencing primers strand,wherein the forward extension strand is hybridized to the immobilizedconcatemer template molecule (FIG. 63 ). For example, one of theextended forward sequencing primer strands can serve as a primer for thestrand displacing polymerase. The strand displacing polymerase canextend the extended forward sequencing primer strand, and displacedownstream extended forward sequencing primer strands while synthesizingan extended strand that replaces the downstream extended forwardsequencing primer strands. The newly extended strand is covalentlyjoined to an extended forward sequencing primer strand. The immobilizedconcatemer template molecules are retained. The primer extensionreaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (f) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble forward sequencing primers (e.g., a second plurality ofsoluble forward sequencing primers), a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble forward sequencing primers to the plurality of retainedimmobilized concatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the soluble sequencingprimers hybridize with the forward sequencing primer binding sequence inthe retained immobilized concatemer molecules (FIG. 64 ). The primerextension reaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (f), the condition suitable to hybridizethe plurality of soluble forward sequencing primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, step (f) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of primer extensionpolymerases, under a condition suitable to hybridize the plurality ofsoluble amplification primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conductingpolymerase-catalyzed primer extension reactions thereby generating aplurality of forward extension strands, wherein the solubleamplification primers hybridize with the soluble amplification primerbinding sequence in the retained immobilized concatemer molecules (FIG.65 ). The primer extension reaction can optionally include a pluralityof compaction oligonucleotides and/or hexamine (e.g., cobalt hexamineIII) to generate forward extension strands. Individual forward extensionstrands can collapse into a nanoball having a more compact size and/orshape compared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, in step (f), the condition suitable to hybridizethe plurality of soluble amplification primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing retained immobilized concatemer templatemolecules with the soluble primers in the presence of a primer extensionpolymerase, a plurality of nucleotides, and a high efficiencyhybridization buffer. In some embodiment, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, in step (f), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (f), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or abuffering agent (e.g., Tris-HCl, MES, HEPES, or the like).

In some embodiments, in step (f), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (f), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the primer extension polymerase of step (f)comprises a high fidelity polymerase. In some embodiments, the primerextension polymerase of step (d) comprises a DNA polymerase capable ofcatalyzing a primer extension reaction using a uracil-containingtemplate molecule (e.g., a uracil-tolerant polymerase). Exemplarypolymerases include, but are not limited to, Q5U Hot Start high-fidelityDNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNApolymerase, One Taq DNA polymerase (e.g., mixture of Taq and Deep VentDNA polymerases, catalog #M0480S from New England Biolabs), LongAmp TaqDNA polymerase (e.g., catalog #M0323S from New England Biolabs), EpimarkHot Start Taq DNA polymerase (e.g., catalog #M0490S from New EnglandBiolabs), Bst DNA polymerase (e.g., large fragment, catalog #M0275S fromNew England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog#M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog#M0269S from New England Biolabs), E. coli DNA polymerase (e.g., catalog#M0209S from New England Biolabs), Therminator DNA polymerase (e.g.,catalog #M0261S from New England Biolabs), Vent DNA polymerase and DeepVent DNA polymerase.

The pairwise methods described herein can provide increased accuracy ina downstream sequencing reaction because step (f) replaces the extendedforward sequencing primer strands that were generated in step (e) withforward extension strands having reduced base errors. The extendedforward sequencing primer strands are generated in step (e) and may ormay not contain erroneously incorporated nucleotides due topolymerase-catalyzed mis-paired bases. When step (e) is conducted with ahigh fidelity DNA polymerase, the resulting forward extension strandsmay have reduced base errors compared to the extended forward sequencingprimer strands. The forward extension strands will be used as a nucleicacid template for a downstream sequencing step (e.g., see step (h)below). Thus, step (f) can increase the sequencing accuracy of thedownstream step (h) and therefore increase the overall sequencingaccuracy of the pairwise sequencing workflow.

In some embodiments, the pairwise sequencing method further comprisesstep (g): removing the retained immobilized concatemer templatemolecules by generating abasic sites in the immobilized single strandedconcatemer template molecules at the nucleotide(s) having the scissilemoiety and generating gaps at the abasic sites to generate a pluralityof gap-containing single stranded nucleic acid concatemer templatemolecules while retaining the plurality of forward extension strands andretaining the plurality of immobilized surface primers (FIGS. 66 and 67, and FIGS. 68 and 69 ).

The abasic sites are generated on the retained concatemer templatestrands that contain nucleotides having scissile moieties. In someembodiments, the scissile moieties in the retained concatemer templatemolecules comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine. The abasic sites can be removed to generate a plurality ofsingle stranded nucleic acid template molecules having gaps whileretaining the plurality of forward extension strands. The abasic sitescan be generated by contacting the immobilized concatemer templatemolecules with an enzyme that removes the nucleo-base at the nucleotidehaving the scissile moiety. The uracil in the retained concatemertemplate strands can be converted to an abasic site using uracil DNAglycosylase (UDG). The 8oxoG in the retained concatemer template strandscan be converted to an abasic site using FPG glycosylase. Thedeoxyinosine in the retained concatemer template strands can beconverted to an abasic site using AlkA glycosylase.

In some embodiments, in step (g), the gaps can be generated bycontacting the abasic sites in the immobilized concatemer templatemolecules with an enzyme or a mixture of enzymes having lyase activitythat breaks the phosphodiester backbone at the 5′ and 3′ sides of theabasic site to release the base-free deoxyribose and generate a gap(FIGS. 66 and 68 ). The abasic sites can be removed using AP lyase, EndoIV endonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/APlyase. In some embodiments, generating the abasic sites and removal ofthe abasic sites to generate gaps can be achieved using a mixture ofuracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, forexample USER (Uracil-Specific Excision Reagent Enzyme from New EnglandBiolabs) or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (g), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical compoundand/or heat. After the gap-removal procedure, the plurality of retainedforward extension strands are hybridized to the retained immobilizedsurface primers (FIGS. 67 and 69 ).

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S). When a 5′ to 3′ double-stranded DNA exonuclease is used forremoving gap-containing template molecules, then the plurality ofsoluble amplification primers in step (f) can comprise at least onephosphorothioate diester bond at their 5′ ends which can render thesoluble amplification primers resistant to exonuclease degradation. Insome embodiments, the plurality of soluble amplification primers in step(f) comprise 2-5 or more consecutive phosphorothioate diester bonds attheir 5′ ends. In some embodiments, the plurality soluble amplificationprimers in step (f) comprise at least one ribonucleotide and/or at leastone 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotide which can renderthe forward sequencing primers resistant to exonuclease degradation.

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (h): sequencing the plurality of retained forward extension strandsthereby generating a plurality of extended reverse sequencing primerstrands. In some embodiments, the sequencing of step (h) comprisescontacting the plurality of retained forward extension strands with aplurality of soluble reverse sequencing primers under a conditionsuitable to hybridize the reverse sequencing primers to the reversesequencing primer binding site of the retained forward extensionstrands, and by conducting sequencing reactions using the hybridizedreverse sequencing primers wherein the forward sequencing reactionsgenerates a plurality of extended reverse sequencing primer strands(FIGS. 70 and 71 ). The extended reverse sequencing primer strands arehybridized to the retained forward extension strand. The retainedforward extension strand is hybridized to the first surface primer. Theextended reverse sequencing primer strands are not hybridized to thefirst surface primer, or covalently joined to the first surface primer.Therefore, the extended reverse sequencing primer strands are notimmobilized to the support.

In some embodiments, in step (h), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In an alternative embodiment, the sequencing of step (h) comprises usingthe immobilized surface primer as a sequencing primer and conductingsequencing reactions to generate a plurality of reverse sequencingstrands.

In some embodiments, the reverse sequencing reactions of step (h)comprises contacting the plurality of reverse sequencing primers withthe reverse sequencing primer binding sequences of the retained forwardextension strands, one or more types of sequencing polymerases, and aplurality of nucleotides and/or a plurality of multivalent molecules. Insome embodiments, the soluble reverse sequencing primers comprise 3′ OHextendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules is described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site (e.g., seeFIGS. 70 and 71 ). In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having nucleotide units,where the multivalent molecules are labeled with a detectable reportermoiety. In some embodiments, the detectable reporter moiety comprises afluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(h). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (di ethylene triamine pentaacetic acid), NIA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

Methods for Pairwise Sequencing—Lacking Abasic Sites

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a plurality of immobilized single stranded nucleicacid concatemer template molecules each lacking a scissile moiety thatcan be cleaved to generate an abasic site in the concatemer templatemolecule, wherein individual concatemer template molecules in theplurality are immobilized to a first surface primer that is immobilizedto a support, and wherein the immobilized first surface primer lacks anucleotide having a scissile moiety. In some embodiments, the supportcomprises a plurality of first surface primers. In some embodiments, thesupport lacks a plurality of second surface primers. In someembodiments, the support comprises a plurality of first and secondsurface primers. Exemplary nucleotides having a scissile moiety includeuridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine.

In some embodiments, individual immobilized concatemer templatemolecules are covalently joined to an immobilized surface primer (e.g.,an immobilized first surface primer) (FIG. 73 ). In an alternativeembodiment, individual immobilized concatemer template molecules arehybridized to an immobilized surface primer (e.g., an immobilized firstsurface primer) (FIG. 80 ).

In some embodiments, individual concatemer template molecules in theplurality comprise two or more copies of a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of: (i) two or morecopies of a universal binding sequence for a soluble forward sequencingprimer, (ii) two or more copies of a universal binding sequence for asoluble reverse sequencing primer, (iii) two or more copies of auniversal binding sequence for an immobilized first surface primer, (iv)two or more copies of a universal binding sequence for an immobilizedsecond surface primer, (v) two or more copies of a universal bindingsequence for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence for a second solubleamplification primer, (vii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, individual concatemer template molecules in theplurality comprise two or more copies of a sequence of interest and twoor more copies of a universal binding sequence for a soluble compactionoligonucleotide, and wherein the individual immobilized concatemertemplate molecules further comprise any one or any combination of two ormore of: (i) two or more copies of a universal binding sequence for asoluble forward sequencing primer, (ii) two or more copies of auniversal binding sequence for a soluble reverse sequencing primer,(iii) two or more copies of a universal binding sequence for animmobilized first surface primer, (iv) two or more copies of a universalbinding sequence for an immobilized second surface primer, (v) two ormore copies of a universal binding sequence for a first solubleamplification primer, (vi) two or more copies of a universal bindingsequence for a second soluble amplification primer, (vii) two or morecopies of a sample barcode sequence and/or (viii) two or more copies ofa unique molecular index sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized first surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedfirst surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized first surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a 3′ non-extendible moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the immobilized concatemer template moleculesfurther comprise two or more copies of a universal binding sequence (orcomplementary sequence thereof) for an immobilized second surface primerhaving a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized second surface primers can be immobilizedto the support or immobilized to a coating on the support. Theimmobilized second surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized second surface primers are immobilized to a supportor immobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise a moietythat blocks primer extension (e.g., non-extendible terminal 3′ end),such as for example a phosphate group, a dideoxycytidine group, aninverted dT, or an amino group. The immobilized second surface primersare not extendible in a primer extension reaction. The immobilizedsecond surface primers lack a nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecules are joined or immobilized to animmobilized first surface primer, and at least one portion of theindividual concatemer template molecule is hybridized to an immobilizedsecond surface primer. The immobilized second surface primers serve topin down a portion of the immobilized concatemer template molecules tothe support (see FIGS. 79 and 86 ).

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (b): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (b) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers. In some embodiments, the soluble forwardsequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble forward sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble forward sequencing primers lack a nucleotidehaving a scissile moiety. The forward sequencing reactions can generatea plurality of extended forward sequencing primer strands. In someembodiments, individual immobilized concatemer template molecules havemultiple copies of the forward sequencing primer binding sites, whereineach forward sequencing primer binding site is capable of hybridizing toa first forward sequencing primer. Individual forward sequencing primerbinding sites in a given immobilized concatemer template molecule can behybridized to a forward sequencing primer and can undergo a sequencingreaction. Individual immobilized concatemer template molecules canundergo two or more sequence reactions, where each sequencing reactionis initiated from a first forward sequencing primer that is hybridizedto a forward sequencing primer binding site (e.g., see FIGS. 74 and 81). In some embodiments, the sequencing reactions comprise a plurality ofnucleotides (or analogs thereof) labeled with a detectable reportermoiety. In some embodiments, the sequencing reaction comprise aplurality of multivalent molecules having a plurality of nucleotideunits attached to a core, where the multivalent molecules are labeledwith a detectable reporter moiety. In some embodiments, the core islabeled with a detectable reporter moiety. In some embodiments, at leastone linker and/or at least one nucleotide unit of a nucleotide arm islabeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (c): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands byconducting a primer extension reaction. The extended forward sequencingprimer strands can be removed from the retained immobilized concatemertemplate molecules. The retained immobilized concatemer templatemolecule can be hybridized to a plurality of soluble amplification orsequencing primers and subjected to a primer extension reaction. Theprimer extension reaction can be conducted with a plurality of solubleprimers (e.g., soluble amplification primers or soluble forwardsequencing primers) and a plurality of strand-displacing polymerases togenerate a plurality of forward extension strands that are hybridized tothe immobilized concatemer template molecules, and a plurality ofpartially displaced forward extension strands that are hybridized to theimmobilized concatemer template molecules to form a plurality ofimmobilized amplicons. The strand displacing primer extension reactionalso generate a plurality of detached forward extension strands that arenot hybridized to the immobilized concatemer template molecules. In someembodiments, the strand displacing primer extension reaction can beconducted in the presence of a plurality of soluble compactionoligonucleotides to immobilize the detached forward extension strands tothe immobilized amplicons (see FIGS. 75-77 and FIGS. 82-84 ).

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (c) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of strand displacingpolymerases, under a condition suitable to hybridize the plurality ofsoluble amplification primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conducting a stranddisplacing primer extension reactions thereby generating a plurality offorward extension strands and a plurality of partially displaced forwardextension strands that are hybridized to the immobilized concatemertemplate molecules, and a plurality of detached forward extensionstrands that are not hybridized to the immobilized concatemer templatemolecules. The soluble amplification primers hybridize with the solubleamplification primer binding sequence in the retained immobilizedconcatemer molecules (FIGS. 75 and 82 ). The primer extension reactioncan optionally include a plurality of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) to generate forward extensionstrands. Individual forward extension strands can collapse into ananoball having a more compact size and/or shape compared to a nanoballgenerated from a primer extension reaction conducted without compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusionof compaction oligonucleotides and/or hexamine (e.g., cobalt hexamineIII) in the primer extension reaction can improve FWHM (full width halfmaximum) of a spot image of the nanoball. The spot image can berepresented as a Gaussian spot and the size can be measured as a FWHM. Asmaller spot size as indicated by a smaller FWHM typically correlateswith an improved image of the spot. In some embodiments, the FWHM of ananoball spot can be about 10 μm or smaller.

In some embodiments, step (c) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble sequencing primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of strand displacingpolymerases, under a condition suitable to hybridize the plurality ofsoluble sequencing primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conducting a stranddisplacing primer extension reactions thereby generating a plurality offorward extension strands and a plurality of partially displaced forwardextension strands that are hybridized to the immobilized concatemertemplate molecules, and a plurality of detached forward extensionstrands that are not hybridized to the immobilized concatemer templatemolecules. The soluble forward sequencing primers hybridize with theforward sequencing primer binding sequence in the retained immobilizedconcatemer molecules. The primer extension reaction can optionallyinclude a plurality of compaction oligonucleotides and/or hexamine(e.g., cobalt hexamine III) to generate forward extension strands.Individual forward extension strands can collapse into a nanoball havinga more compact size and/or shape compared to a nanoball generated from aprimer extension reaction conducted without compaction oligonucleotidesand/or hexamine (e.g., cobalt hexamine III). Inclusion of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) in theprimer extension reaction can improve FWHM (full width half maximum) ofa spot image of the nanoball. The spot image can be represented as aGaussian spot and the size can be measured as a FWHM. A smaller spotsize as indicated by a smaller FWHM typically correlates with animproved image of the spot. In some embodiments, the FWHM of a nanoballspot can be about 10 μm or smaller.

In some embodiments, in step (c), the condition suitable to hybridizethe plurality of soluble amplification primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing the retained immobilized concatemertemplate molecules with the soluble amplification primers in thepresence of a primer extension polymerase, a plurality of nucleotides,and a high efficiency hybridization buffer. In some embodiment, the highefficiency hybridization buffer comprises: (i) a first polar aproticsolvent having a dielectric constant that is no greater than 40 andhaving a polarity index of 4-9; (ii) a second polar aprotic solventhaving a dielectric constant that is no greater than 115 and is presentin the hybridization buffer formulation in an amount effective todenature double-stranded nucleic acids; (iii) a pH buffer system thatmaintains the pH of the hybridization buffer formulation in a range ofabout 4-8; and (iv) a crowding agent in an amount sufficient to enhanceor facilitate molecular crowding. In some embodiments, the highefficiency hybridization buffer comprises: (i) the first polar aproticsolvent comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) the second polar aprotic solvent comprises formamide at5-10% by volume of the hybridization buffer; (iii) the pH buffer systemcomprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5;and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35%by volume of the hybridization buffer. In some embodiments, the highefficiency hybridization buffer further comprises betaine.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (c), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (d): sequencing the plurality of immobilized partially displacedforward extension strands thereby generating a first plurality ofextended reverse sequencing primer strands. In some embodiments, step(d) further comprises sequencing the plurality of immobilized detachedforward extension strands thereby generating a second plurality ofextended reverse sequencing primer strands. In some embodiments,individual immobilized partially displaced forward extension strandshave two or more extended reverse sequencing primer strands hybridizedthereon. In some embodiments, individual immobilized detached forwardextension strands have two or more extended reverse sequencing primerstrands hybridized thereon. During the sequencing of step (d), theimmobilized partially displaced forward extension strands remainhybridized to the retained immobilized concatemer template molecules.

In some embodiments, the sequencing of step (d) comprises contacting theplurality of immobilized partially displaced forward extension strands(e.g., that are hybridized to the immobilized concatemer templatemolecules), and the plurality of immobilized detached forward extensionstrands, with a plurality of soluble reverse sequencing primers under acondition suitable to hybridize the reverse sequencing primers to thereverse sequencing primer binding site of the forward extension strands(FIGS. 78 and 85 ). The sequencing of step (d) comprises conductingsequencing reactions using the hybridized reverse sequencing primerswherein the forward sequencing reactions generates a plurality ofextended reverse sequencing primer strands (FIGS. 78 and 85 ). Theextended reverse sequencing primer strands are hybridized to a partiallydisplaced forward extension strand that is hybridized to the immobilizedconcatemer template molecules, or an immobilized detached forwardextension strand.

For the sake of simplicity, FIGS. 73-78 and 80-85 do not show animmobilized concatemer template molecule having a universal bindingsequence for a soluble compaction oligonucleotide. The skilled artisanwill appreciate that the immobilized concatemer template molecule caninclude a universal binding sequence for a soluble compactionoligonucleotide.

For the sake of simplicity, FIGS. 78 and 85 show an exemplaryimmobilized partially displaced forward extension strand that ishybridized to the immobilized concatemer template molecule, and theimmobilized detached forward extension strand, each having one copy ofan extended reverse sequencing primer strand hybridized thereon. Theskilled artisan will appreciate that the immobilized partially displacedforward extension strand that is hybridized to the immobilizedconcatemer template molecule, and the immobilized detached forwardextension strand, can have two or more copies of the extended reversesequencing primer strands hybridized thereon. Therefore, the reversesequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same immobilized partiallydisplaced forward extension strand that is hybridized to the immobilizedconcatemer template molecule, or the immobilized detached forwardextension strand.

In some embodiments, the reverse sequencing reaction can include aplurality of compaction oligonucleotides. The compactionoligonucleotides can serve to immobilize one or more of the detachedforward extension strands via hybridization to the immobilized partiallydisplaced forward extension strand (e.g., that is hybridized to theimmobilized concatemer template molecule).

In some embodiments, in step (d), the condition that is suitable tohybridize the reverse sequencing primers to the reverse sequencingprimer binding sequences of the immobilized partially displaced forwardextension strand that is hybridized to the immobilized concatemertemplate molecule, and the immobilized detached forward extensionstrand, comprises contacting the plurality of soluble reverse sequencingprimers and the forward extension strands with a high efficiencyhybridization buffer. In some embodiments, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reactions of step (d)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the immobilizedpartially displaced forward extension strand that is hybridized to theimmobilized concatemer template molecule, or the immobilized detachedforward extension strand, with one or more types of sequencingpolymerases, and a plurality of nucleotides and/or a plurality ofmultivalent molecules. In some embodiments, the soluble reversesequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble reverse sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble reverse sequencing primers lack a nucleotidehaving a scissile moiety. The sequencing reactions that employnucleotides and/or multivalent molecules is described in more detailbelow. The reverse sequencing reactions can generate a plurality ofextended reverse sequencing primer strands. In some embodiments,individual forward extension strands have multiple copies of the reversesequencing primer binding sequences/sites, wherein each reversesequencing primer binding site is capable of hybridizing to a reversesequencing primer. Individual reverse sequencing primer binding sites ina given immobilized partially displaced forward extension strand that ishybridized to the immobilized concatemer template molecule, orimmobilized detached forward extension strand, can be hybridized to areverse sequencing primer and can undergo a sequencing reaction. Thus,an individual retained forward extension strand can undergo two or moresequence reactions, where each sequencing reaction is initiated from areverse sequencing primer that is hybridized to a reverse sequencingprimer binding site. In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having nucleotide units,where the multivalent molecules are labeled with a detectable reportermoiety. In some embodiments, the detectable reporter moiety comprises afluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(d). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic add), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

On Support RCA and Pairwise Sequencing—Lacking Abasic Sites

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a support having a plurality of surface primers(e.g., a plurality of first surface primers) immobilized thereon whereineach of the surface primers have a 3′ OH extendible end and lack anucleotide having a scissile moiety (FIG. 87 ). For example, the surfaceprimers lack uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) anddeoxyinosine. In some embodiments, the support comprises a plurality offirst surface primers. In some embodiments, the support lacks aplurality of second surface primers. In some embodiments, the supportcomprises a plurality of first and second surface primers.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The first surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of a nucleic acid library molecule (e.g., linear orcircular library molecules). The first surface primers can include aterminal 3′ nucleotide having a sugar 3′ OH moiety which is extendiblefor nucleotide polymerization (e.g., polymerase catalyzedpolymerization).

The immobilized first surface primers can be immobilized to the supportor immobilized to a coating on the support. The immobilized firstsurface primers can be embedded and attached (coupled) to the coating onthe support. In some embodiments, the 5′ end of the immobilized firstsurface primers are immobilized to a support or immobilized to a coatingon the support. Alternatively, an interior portion or the 3′ end of theimmobilized first surface primers can be immobilized to a support orimmobilized to a coating on the support. The support comprises aplurality of immobilized first surface primers having the same sequence.The immobilized first surface primers can be any length, for example4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides, orlonger lengths.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the support further comprises a plurality of asecond surface primer immobilized thereon (e.g., FIG. 79 ). The secondsurface primers have a sequence that differs from the first immobilizedsurface primer. The immobilized second surface primers of step (a)comprise single stranded oligonucleotides comprising DNA, RNA or acombination of DNA and RNA. The second surface primers comprise asequence that is wholly complementary or partially complementary alongtheir lengths to at least a portion of an immobilized single strandedconcatemer template molecule. The immobilized second surface primers canbe immobilized to the support or immobilized to a coating on thesupport. The immobilized second surface primers can be embedded andattached (coupled) to the coating on the support. In some embodiments,the 5′ end of the immobilized second surface primers are immobilized toa support or immobilized to a coating on the support. Alternatively, aninterior portion or the 3′ end of the immobilized second surface primerscan be immobilized to a support or immobilized to a coating on thesupport. The support comprises a plurality of immobilized second surfaceprimers having the same sequence. The immobilized second surface primerscan be any length, for example 4-50 nucleotides, or 50-100 nucleotides,or 100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized second surface primers comprise a 3′ non-extendible moiety.The 3′ terminal end of the immobilized second surface primers comprise amoiety that blocks primer extension, such as for example a phosphategroup, a dideoxycytidine group, an inverted dT, or an amino group. Theimmobilized second surface primers are not extendible in a primerextension reaction. The immobilized second surface primers lack anucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are covalently joined to an immobilizedfirst surface primer, and at least one portion of the individualconcatemer template molecule is hybridized to an immobilized secondsurface primer (e.g., FIG. 79 ). The immobilized second surface primersserve to pin down a portion of the immobilized concatemer templatemolecules to the support. The immobilized concatemer template moleculehas two or more copies of a universal binding sequence for animmobilized second surface primer. The portion of the immobilizedconcatemer template molecule that includes the universal bindingsequence for an immobilized second surface primer can hybridize to theimmobilized second surface primer. In some embodiments, the secondsurface primers include a terminal 3′ blocking group that renders themnon-extendible. In some embodiments, the second surface primers haveterminal 3′ extendible ends.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

The present disclosure provides pairwise sequencing methods, comprisingstep (b): generating a plurality of immobilized single stranded nucleicacid concatemer template molecules by hybridizing a plurality ofsingle-stranded circular nucleic acid library molecules to the pluralityof immobilized first surface primers and conducting a rolling circleamplification reaction. In some embodiments, the rolling circleamplification reaction can be conducted with a plurality of a stranddisplacing polymerase, and a plurality of nucleotides which lack anucleotide having a scissile moiety that can be cleaved to generate anabasic site. In some embodiments, the plurality of nucleotides comprisesany combination of dATP, dCTP, dGTP and/or dTTP. The rolling circleamplification reaction includes a mixture of nucleotides that lack ascissile moiety. Exemplary nucleotides having a scissile moiety includeuridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine. Insome embodiments, the rolling circle amplification reaction generates aplurality of immobilized single stranded nucleic acid concatemertemplate molecules that lack a nucleotide having a scissile moiety,wherein individual single stranded nucleic acid concatemer templatemolecules are covalently joined to an immobilized first surface primer(FIG. 88 ).

In some embodiments, the single-stranded circular nucleic acid librarymolecules comprise covalently closed circular molecules. In someembodiments, the single-stranded circular nucleic acid library moleculescan be removed from the concatemer template molecules with at least onewashing step which is conducted under a condition suitable to retain thesingle stranded nucleic acid concatemer template molecules whereindividual concatemer template molecules are operably joined to animmobilized first surface primer.

In some embodiments, individual single stranded circular nucleic acidlibrary molecules in the plurality comprise a sequence of interest, andthe individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) a universalbinding sequence (or complementary sequence thereof) for a solubleforward sequencing primer, (ii) a universal binding sequence (orcomplementary sequence thereof) for a soluble reverse sequencing primer,(iii) a universal binding sequence (or complementary sequence thereof)for an immobilized first surface primer, (iv) a universal bindingsequence (or complementary sequence thereof) for an immobilized secondsurface primer, (v) a universal binding sequence (or complementarysequence thereof) for a first soluble amplification primer, (vi) auniversal binding sequence (or complementary sequence thereof) for asecond soluble amplification primer, (vii) a universal binding sequence(or complementary sequence thereof) for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual single stranded circular nucleic acidlibrary molecules in the plurality comprise a sequence of interest and auniversal binding sequence (or complementary sequence thereof) for asoluble compaction oligonucleotide, and the individual immobilizedconcatemer template molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence (orcomplementary sequence thereof) for a soluble forward sequencing primer,(ii) a universal binding sequence (or complementary sequence thereof)for a soluble reverse sequencing primer, (iii) a universal bindingsequence (or complementary sequence thereof) for an immobilized firstsurface primer, (iv) a universal binding sequence (or complementarysequence thereof) for an immobilized second surface primer, (v) auniversal binding sequence (or complementary sequence thereof) for afirst soluble amplification primer, (vi) a universal binding sequence(or complementary sequence thereof) for a second soluble amplificationprimer, (vii) a sample barcode sequence and/or (viii) a unique molecularindex sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the rolling circle amplification reaction of step(b) generates a plurality of immobilized single stranded nucleic acidconcatemer template molecules each comprising a concatemer lacking anucleotide having a scissile moiety and two or more copies of a sequenceof interest, and wherein the immobilized concatemer template moleculesfurther comprise any one or any combination of two or more of: (i) twoor more copies of a universal binding sequence (or a complementarysequence thereof) for a soluble forward sequencing primer, (ii) two ormore copies of a universal binding sequence (or a complementary sequencethereof) for a soluble reverse sequencing primer, (iii) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for an immobilized first surface primer, (iv) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for an immobilized second surface primer, (v) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for a second soluble amplification primer, (vii) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, the plurality of immobilized single strandednucleic acid concatemer template molecules that are generated by therolling circle amplification reaction of step (b) further comprise twoor more copies of a universal binding sequence (or complementarysequence thereof) for immobilized second sequence surface primers. Insome embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are joined (e.g., covalently joined) to animmobilized first surface primer, and at least one portion of theindividual concatemer template molecule is hybridized to an immobilizedsecond surface primer. The immobilized second surface primers serve topin down a portion of the immobilized concatemer template molecules tothe support (e.g., see FIG. 79 ). In some embodiments, the secondsurface primers include a terminal 3′ blocking group that renders themnon-extendible.

The rolling circle amplification reaction of step (b) can be conductedwith a nucleotide mixture containing dATP, dCTP, dGTP, dTTP, and thenucleotide mixture lacks a nucleotide having a scissile moiety.Exemplary nucleotides having a scissile moiety includes uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine.

In some embodiments, the pairwise sequencing method further comprisesstep (c): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (c) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers. The forward sequencing reactions cangenerate a plurality of extended forward sequencing primer strands. Insome embodiments, individual immobilized concatemer template moleculeshave multiple copies of the forward sequencing primer binding sites,wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site(e.g., see FIG. 90 ). In some embodiments, the soluble forwardsequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble forward sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble forward sequencing primers lack a nucleotidehaving a scissile moiety. In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having a plurality ofnucleotide units attached to a core, where the multivalent molecules arelabeled with a detectable reporter moiety. In some embodiments, the coreis labeled with a detectable reporter moiety. In some embodiments, atleast one linker and/or at least one nucleotide unit of a nucleotide armis labeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (d): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands byconducting a primer extension reaction. The extended forward sequencingprimer strands can be removed from the retained immobilized concatemertemplate molecules. The retained immobilized concatemer templatemolecule can be hybridized to a plurality of soluble amplification orsequencing primers and subjected to a primer extension reaction. Theprimer extension reaction can be conducted with a plurality of solubleprimers (e.g., soluble amplification primers or soluble forwardsequencing primers) and a plurality of strand-displacing polymerases togenerate a plurality of forward extension strands that are hybridized tothe immobilized concatemer template molecules, and a plurality ofpartially displaced forward extension strands that are hybridized to theimmobilized concatemer template molecules to form a plurality ofimmobilized amplicons. The strand displacing primer extension reactionalso generate a plurality of detached forward extension strands that arenot hybridized to the immobilized concatemer template molecules. In someembodiments, the strand displacing primer extension reaction can beconducted in the presence of a plurality of soluble compactionoligonucleotides to immobilize the detached forward extension strands tothe immobilized amplicons (see FIGS. 91-93 ).

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (d) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble amplification primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of strand displacingpolymerases, under a condition suitable to hybridize the plurality ofsoluble amplification primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conducting a stranddisplacing primer extension reactions thereby generating a plurality offorward extension strands and a plurality of partially displaced forwardextension strands that are hybridized to the immobilized concatemertemplate molecules, and a plurality of detached forward extensionstrands that are not hybridized to the immobilized concatemer templatemolecules. The soluble amplification primers hybridize with the solubleamplification primer binding sequence in the retained immobilizedconcatemer molecules (FIG. 91 ). The primer extension reaction canoptionally include a plurality of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) to generate forward extensionstrands. Individual forward extension strands can collapse into ananoball having a more compact size and/or shape compared to a nanoballgenerated from a primer extension reaction conducted without compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusionof compaction oligonucleotides and/or hexamine (e.g., cobalt hexamineIII) in the primer extension reaction can improve FWHM (full width halfmaximum) of a spot image of the nanoball. The spot image can berepresented as a Gaussian spot and the size can be measured as a FWHM. Asmaller spot size as indicated by a smaller FWHM typically correlateswith an improved image of the spot. In some embodiments, the FWHM of ananoball spot can be about 10 μm or smaller.

In some embodiments, step (d) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof soluble sequencing primers, a plurality of nucleotides (e.g., asecond plurality of nucleotides) and a plurality of strand displacingpolymerases, under a condition suitable to hybridize the plurality ofsoluble sequencing primers to the plurality of retained immobilizedconcatemer template molecules and suitable for conducting a stranddisplacing primer extension reactions thereby generating a plurality offorward extension strands and a plurality of partially displaced forwardextension strands that are hybridized to the immobilized concatemertemplate molecules, and a plurality of detached forward extensionstrands that are not hybridized to the immobilized concatemer templatemolecules. The soluble forward sequencing primers hybridize with theforward sequencing primer binding sequence in the retained immobilizedconcatemer molecules. The primer extension reaction can optionallyinclude a plurality of compaction oligonucleotides and/or hexamine(e.g., cobalt hexamine III) to generate forward extension strands.Individual forward extension strands can collapse into a nanoball havinga more compact size and/or shape compared to a nanoball generated from aprimer extension reaction conducted without compaction oligonucleotidesand/or hexamine (e.g., cobalt hexamine III). Inclusion of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) in theprimer extension reaction can improve FWHM (full width half maximum) ofa spot image of the nanoball. The spot image can be represented as aGaussian spot and the size can be measured as a FWHM. A smaller spotsize as indicated by a smaller FWHM typically correlates with animproved image of the spot. In some embodiments, the FWHM of a nanoballspot can be about 10 μm or smaller.

In some embodiments, in step (d), the condition suitable to hybridizethe plurality of soluble amplification primers to the plurality ofretained immobilized single stranded nucleic acid concatemer templatemolecules comprises hybridizing the retained immobilized concatemertemplate molecules with the soluble amplification primers in thepresence of a primer extension polymerase, a plurality of nucleotides,and a high efficiency hybridization buffer. In some embodiment, the highefficiency hybridization buffer comprises: (i) a first polar aproticsolvent having a dielectric constant that is no greater than 40 andhaving a polarity index of 4-9; (ii) a second polar aprotic solventhaving a dielectric constant that is no greater than 115 and is presentin the hybridization buffer formulation in an amount effective todenature double-stranded nucleic acids; (iii) a pH buffer system thatmaintains the pH of the hybridization buffer formulation in a range ofabout 4-8; and (iv) a crowding agent in an amount sufficient to enhanceor facilitate molecular crowding. In some embodiments, the highefficiency hybridization buffer comprises: (i) the first polar aproticsolvent comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) the second polar aprotic solvent comprises formamide at5-10% by volume of the hybridization buffer; (iii) the pH buffer systemcomprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5;and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35%by volume of the hybridization buffer. In some embodiments, the highefficiency hybridization buffer further comprises betaine.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (d), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (e): sequencing the plurality of immobilized partially displacedforward extension strands thereby generating a first plurality ofextended reverse sequencing primer strands. In some embodiments, step(e) further comprises sequencing the plurality of immobilized detachedforward extension strands thereby generating a second plurality ofextended reverse sequencing primer strands. In some embodiments,individual immobilized partially displaced forward extension strandshave two or more extended reverse sequencing primer strands hybridizedthereon. In some embodiments, individual immobilized detached forwardextension strands have two or more extended reverse sequencing primerstrands hybridized thereon. During the sequencing of step (e), theimmobilized partially displaced forward extension strands remainhybridized to the retained immobilized concatemer template molecules.

In some embodiments, the sequencing of step (e) comprises contacting theplurality of immobilized partially displaced forward extension strands(e.g., that are hybridized to the immobilized concatemer templatemolecules), and the plurality of immobilized detached forward extensionstrands, with a plurality of soluble reverse sequencing primers under acondition suitable to hybridize the reverse sequencing primers to thereverse sequencing primer binding site of the forward extension strands(FIG. 94 ). The sequencing of step (e) comprises conducting sequencingreactions using the hybridized reverse sequencing primers wherein thereverse sequencing reactions generates a plurality of extended reversesequencing primer strands (FIG. 94 ). The extended reverse sequencingprimer strands are hybridized to a partially displaced forward extensionstrand that is hybridized to the immobilized concatemer templatemolecules, or an immobilized detached forward extension strand.

For the sake of simplicity, FIGS. 88-94 do not show an immobilizedconcatemer template molecule having a universal binding sequence for asoluble compaction oligonucleotide. The skilled artisan will appreciatethat the immobilized concatemer template molecule can include auniversal binding sequence for a soluble compaction oligonucleotide.

For the sake of simplicity, FIG. 94 shows an exemplary immobilizedpartially displaced forward extension strand that is hybridized to theimmobilized concatemer template molecule, and the immobilized detachedforward extension strand, each having one copy of an extended reversesequencing primer strand hybridized thereon. The skilled artisan willappreciate that the immobilized partially displaced forward extensionstrand that is hybridized to the immobilized concatemer templatemolecule, and the immobilized detached forward extension strand, canhave two or more copies of the extended reverse sequencing primerstrands hybridized thereon. Therefore, the reverse sequencing reactioncan generate a plurality of extended reverse sequencing primer strandshybridized to the same immobilized partially displaced forward extensionstrand that is hybridized to the immobilized concatemer templatemolecule, or the immobilized detached forward extension strand.

In some embodiments, the reverse sequencing reaction can include aplurality of compaction oligonucleotides. The compactionoligonucleotides can serve to immobilize one or more of the detachedforward extension strands via hybridization to the immobilized partiallydisplaced forward extension strand (e.g., that is hybridized to theimmobilized concatemer template molecule).

In some embodiments, in step (e), the condition that is suitable tohybridize the reverse sequencing primers to the reverse sequencingprimer binding sequences of the immobilized partially displaced forwardextension strand that is hybridized to the immobilized concatemertemplate molecule, and the immobilized detached forward extensionstrand, comprises contacting the plurality of soluble reverse sequencingprimers and the forward extension strands with a high efficiencyhybridization buffer. In some embodiments, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reactions of step (e)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the immobilizedpartially displaced forward extension strand that is hybridized to theimmobilized concatemer template molecule, or the immobilized detachedforward extension strand, with one or more types of sequencingpolymerases, and a plurality of nucleotides and/or a plurality ofmultivalent molecules. In some embodiments, the soluble reversesequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble reverse sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble reverse sequencing primers lack a nucleotidehaving a scissile moiety. The sequencing reactions that employnucleotides and/or multivalent molecules is described in more detailbelow. The reverse sequencing reactions can generate a plurality ofextended reverse sequencing primer strands. In some embodiments,individual forward extension strands have multiple copies of the reversesequencing primer binding sequences/sites, wherein each reversesequencing primer binding site is capable of hybridizing to a reversesequencing primer. Individual reverse sequencing primer binding sites ina given immobilized partially displaced forward extension strand that ishybridized to the immobilized concatemer template molecule, orimmobilized detached forward extension strand, can be hybridized to areverse sequencing primer and can undergo a sequencing reaction. Thus,an individual retained forward extension strand can undergo two or moresequence reactions, where each sequencing reaction is initiated from areverse sequencing primer that is hybridized to a reverse sequencingprimer binding site. In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having nucleotide units,where the multivalent molecules are labeled with a detectable reportermoiety. In some embodiments, the detectable reporter moiety comprises afluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(e). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

In Solution RCA and Pairwise Sequencing—Lacking Abasic Sites

The present disclosure provides pairwise sequencing methods, comprisingstep (a): contacting in-solution a plurality of single-stranded circularnucleic acid library molecules to a plurality of soluble firstamplification primers, a plurality of a strand displacing polymerase,and a plurality of nucleotides which include any combination of dATP,dCTP, dGTP and/or dTTP, under a condition suitable to form a pluralityof library-primer duplexes and suitable for conducting a rolling circleamplification reaction, thereby generating a plurality of singlestranded nucleic acid concatemers (FIG. 95 ). The rolling circleamplification reaction includes a mixture of nucleotides that lack ascissile moiety that can be cleaved to generate an abasic site.Exemplary nucleotides having a scissile moiety include uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine. The rollingcircle amplification reaction generates a plurality of single strandednucleic acid concatemers that lack a nucleotide having a scissilemoiety. In some embodiments, the single-stranded circular nucleic acidlibrary molecules comprise covalently closed circular molecules. In someembodiments, the soluble first amplification primer comprises a sequencethat selectively hybridizes to a universal binding sequence in thecircular nucleic acid library molecules, such as for example a universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer. Alternatively, the soluble firstamplification primer comprises a random sequence that bindsnon-selectively to a sequence in the circular nucleic acid librarymolecules.

In some embodiments, individual single stranded circular nucleic acidlibrary molecules in the plurality comprise a sequence of interest, andthe individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) a universalbinding sequence (or complementary sequence thereof) for a solubleforward sequencing primer, (ii) a universal binding sequence (orcomplementary sequence thereof) for a soluble reverse sequencing primer,(iii) a universal binding sequence (or complementary sequence thereof)for an immobilized first surface primer, (iv) a universal bindingsequence (or complementary sequence thereof) for an immobilized secondsurface primer, (v) a universal binding sequence (or complementarysequence thereof) for a first soluble amplification primer, (vi) auniversal binding sequence (or complementary sequence thereof) for asecond soluble amplification primer, (vii) a universal binding sequence(or complementary sequence thereof) for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, individual single stranded circular nucleic acidlibrary molecules in the plurality comprise a sequence of interest and auniversal binding sequence (or complementary sequence thereof) for asoluble compaction oligonucleotide, and the individual immobilizedconcatemer template molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence (orcomplementary sequence thereof) for a soluble forward sequencing primer,(ii) a universal binding sequence (or complementary sequence thereof)for a soluble reverse sequencing primer, (iii) a universal bindingsequence (or complementary sequence thereof) for an immobilized firstsurface primer, (iv) a universal binding sequence (or complementarysequence thereof) for an immobilized second surface primer, (v) auniversal binding sequence (or complementary sequence thereof) for afirst soluble amplification primer, (vi) a universal binding sequence(or complementary sequence thereof) for a second soluble amplificationprimer, (vii) a sample barcode sequence and/or (viii) a unique molecularindex sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the rolling circle amplification reaction of step(a) generates a plurality of immobilized single stranded nucleic acidconcatemer template molecules each comprising a concatemer lacking anucleotide having a scissile moiety and two or more copies of a sequenceof interest, and wherein the immobilized concatemer template moleculesfurther comprise any one or any combination of two or more of: (i) twoor more copies of a universal binding sequence (or a complementarysequence thereof) for a soluble forward sequencing primer, (ii) two ormore copies of a universal binding sequence (or a complementary sequencethereof) for a soluble reverse sequencing primer, (iii) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for an immobilized first surface primer, (iv) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for an immobilized second surface primer, (v) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for a second soluble amplification primer, (vii) two or morecopies of a universal binding sequence (or a complementary sequencethereof) for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, the pairwise sequencing method further comprisesstep (b): distributing the rolling circle amplification reaction fromstep (a) onto a support having a plurality of the first surface primersimmobilized thereon, under a condition suitable for hybridizing one ormore portions of individual single stranded concatemers to one or moreimmobilized first surface primers (FIG. 96 ). In some embodiments, theimmobilized first surface primers have terminal 3′ group that arenon-extendible. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a moiety that blocks primerextension, such as for example a phosphate group, a dideoxycytidinegroup, an inverted dT, or an amino group. In some embodiments, theimmobilized first surface primer have an extendible 3′OH end. In someembodiments, the immobilized first surface primers lack a nucleotidehaving a scissile moiety. Exemplary nucleotides having a scissile moietyinclude uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) anddeoxyinosine. The concatemers are immobilized to the support byhybridization to the immobilized first surface primers. In someembodiments, the support comprises a plurality of first surface primers.In some embodiments, the support lacks a plurality of second surfaceprimers. In some embodiments, the support comprises a plurality of firstand second surface primers.

In some embodiments, the pairwise sequencing method further comprisesstep (c): continuing the rolling circle amplification reaction on thesupport to generate a plurality of extended concatemer templatemolecules that are immobilized via hybridization to the immobilizedfirst surface primers (FIG. 97 ). The on-support RCA reaction can beconducted with a plurality of a strand displacing polymerase, and aplurality of nucleotides which include any combination of dATP, dCTP,dGTP and/or dTTP. The plurality of nucleotides lack a nucleotide havinga scissile moiety. In some embodiments, the rolling circle amplificationreaction on the support can be conducted in the presence of a pluralityof compaction oligonucleotides.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The first surface primers comprise a sequence that iswholly complementary or partially complementary along their lengths toat least a portion of the concatemer molecules. In some embodiments, thefirst surface primers can lack a terminal 3′ OH extendible end whichrenders the first surface primers non-extendible. In some embodiments,the first surface primers include a terminal 3′ OH group which isextendible for nucleotide polymerization (e.g., polymerase catalyzedpolymerization). The immobilized first surface primers can beimmobilized to the support or immobilized to a coating on the support.The immobilized first surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the plurality of immobilized first surface primerscomprise 3′ extendible ends. In some embodiments, the 3′ terminal end ofthe immobilized first surface primers comprise a moiety that blocksprimer extension, such as for example a phosphate group, adideoxycytidine group, an inverted dT, or an amino group. In someembodiments, the immobilized first surface primers are not extendible ina primer extension reaction. The immobilized first surface primers lacka nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the concatemer template molecules.

In some embodiments, the support further comprises a plurality of asecond surface primer immobilized thereon (FIG. 86 ). The second surfaceprimers have a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers comprise single strandedoligonucleotides comprising DNA, RNA or a combination of DNA and RNA.The second surface primers comprise a sequence that is whollycomplementary or partially complementary along their lengths to at leasta portion of a concatemer molecule. The immobilized second surfaceprimers can be immobilized to the support or immobilized to a coating onthe support. The immobilized second surface primers can be embedded andattached (coupled) to the coating on the support. In some embodiments,the 5′ end of the immobilized second surface primers are immobilized toa support or immobilized to a coating on the support. Alternatively, aninterior portion or the 3′ end of the immobilized second surface primerscan be immobilized to a support or immobilized to a coating on thesupport. The support comprises a plurality of immobilized second surfaceprimers having the same sequence. The immobilized second surface primerscan be any length, for example 4-50 nucleotides, or 50-100 nucleotides,or 100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. The 3′ terminal end of theimmobilized second surface primers comprise a moiety that blocks primerextension, such as for example a phosphate group, a dideoxycytidinegroup, an inverted dT, or an amino group. The immobilized second surfaceprimers are not extendible in a primer extension reaction. Theimmobilized second surface primers lack a nucleotide having a scissilemoiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, individual immobilized single stranded nucleic acidconcatemer template molecule are hybridized to an immobilized firstsurface primer, and at least one portion of the individual concatemertemplate molecule is hybridized to an immobilized second surface primer(FIG. 86 ). The immobilized second surface primers serve to pin down aportion of the immobilized concatemer template molecules to the support.The immobilized concatemer template molecule has two or more copies of auniversal binding sequence for an immobilized second surface primer. Theportion of the immobilized concatemer template molecule that includesthe universal binding sequence for an immobilized second surface primercan hybridize to the immobilized second surface primer. In someembodiments, the second surface primers include a terminal 3′ blockinggroup that renders them non-extendible. In some embodiments, the secondsurface primers have terminal 3′ extendible ends.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers react with the solutions in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (d): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (d) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers. The forward sequencing reactions cangenerate a plurality of extended forward sequencing primer strands. Insome embodiments, individual immobilized concatemer template moleculeshave multiple copies of the forward sequencing primer binding sites,wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site(e.g., see FIG. 99 ). In some embodiments, the soluble forwardsequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble forward sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble forward sequencing primers lack a nucleotidehaving a scissile moiety. In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having a plurality ofnucleotide units attached to a core, where the multivalent molecules arelabeled with a detectable reporter moiety. In some embodiments, the coreis labeled with a detectable reporter moiety. In some embodiments, atleast one linker and/or at least one nucleotide unit of a nucleotide armis labeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 .

In some embodiments, the pairwise sequencing method further comprisesstep (e): retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands byconducting a primer extension reaction. The extended forward sequencingprimer strands can be removed from the retained immobilized concatemertemplate molecules. The retained immobilized concatemer templatemolecule can be hybridized to a plurality of a second solubleamplification primers or a plurality of sequencing primers and subjectedto a primer extension reaction. The primer extension reaction can beconducted with a plurality of soluble primers (e.g., second solubleamplification primers or soluble forward sequencing primers) and aplurality of strand-displacing polymerases to generate a plurality offorward extension strands that are hybridized to the immobilizedconcatemer template molecules, and a plurality of partially displacedforward extension strands that are hybridized to the immobilizedconcatemer template molecules to form a plurality of immobilizedamplicons. The strand displacing primer extension reaction alsogenerates a plurality of detached forward extension strands that are nothybridized to the immobilized concatemer template molecules. In someembodiments, the strand displacing primer extension reaction can beconducted in the presence of a plurality of soluble compactionoligonucleotides to immobilize the detached forward extension strands tothe immobilized amplicons (see FIGS. 100-102 ).

Examples of strand displacing polymerases include phi29 DNA polymerase,large fragment of Bst DNA polymerase, large fragment of Bsu DNApolymerase (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio).

In some embodiments, step (e) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof a second soluble amplification primers, a plurality of nucleotides(e.g., a second plurality of nucleotides) and a plurality of stranddisplacing polymerases, under a condition suitable to hybridize theplurality of second soluble amplification primers to the plurality ofretained immobilized concatemer template molecules and suitable forconducting a strand displacing primer extension reactions therebygenerating a plurality of forward extension strands and a plurality ofpartially displaced forward extension strands that are hybridized to theimmobilized concatemer template molecules, and a plurality of detachedforward extension strands that are not hybridized to the immobilizedconcatemer template molecules. The second soluble amplification primershybridize with the second soluble amplification primer binding sequencein the retained immobilized concatemer molecules (FIG. 100 ). The primerextension reaction can optionally include a plurality of compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generateforward extension strands. Individual forward extension strands cancollapse into a nanoball having a more compact size and/or shapecompared to a nanoball generated from a primer extension reactionconducted without compaction oligonucleotides and/or hexamine (e.g.,cobalt hexamine III). Inclusion of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) in the primer extension reactioncan improve FWHM (full width half maximum) of a spot image of thenanoball. The spot image can be represented as a Gaussian spot and thesize can be measured as a FWHM. A smaller spot size as indicated by asmaller FWHM typically correlates with an improved image of the spot. Insome embodiments, the FWHM of a nanoball spot can be about 10 μm orsmaller.

In some embodiments, step (e) comprises: (i) removing the plurality ofextended forward sequencing primer strand while retaining theimmobilized concatemer template molecules; and (ii) contacting theplurality of retained immobilized concatemer molecules with a pluralityof a second soluble sequencing primers, a plurality of nucleotides(e.g., a second plurality of nucleotides) and a plurality of stranddisplacing polymerases, under a condition suitable to hybridize theplurality of second soluble sequencing primers to the plurality ofretained immobilized concatemer template molecules and suitable forconducting a strand displacing primer extension reactions therebygenerating a plurality of forward extension strands and a plurality ofpartially displaced forward extension strands that are hybridized to theimmobilized concatemer template molecules, and a plurality of detachedforward extension strands that are not hybridized to the immobilizedconcatemer template molecules. The soluble forward sequencing primershybridize with the forward sequencing primer binding sequence in theretained immobilized concatemer molecules. The primer extension reactioncan optionally include a plurality of compaction oligonucleotides and/orhexamine (e.g., cobalt hexamine III) to generate forward extensionstrands. Individual forward extension strands can collapse into ananoball having a more compact size and/or shape compared to a nanoballgenerated from a primer extension reaction conducted without compactionoligonucleotides and/or hexamine (e.g., cobalt hexamine III). Inclusionof compaction oligonucleotides and/or hexamine (e.g., cobalt hexamineIII) in the primer extension reaction can improve FWHM (full width halfmaximum) of a spot image of the nanoball. The spot image can berepresented as a Gaussian spot and the size can be measured as a FWHM. Asmaller spot size as indicated by a smaller FWHM typically correlateswith an improved image of the spot. In some embodiments, the FWHM of ananoball spot can be about 10 μm or smaller.

In some embodiments, in step (e), the condition that is suitable tohybridize the plurality of second soluble amplification primers to theplurality of retained immobilized single stranded nucleic acidconcatemer template molecules comprises hybridizing the retainedimmobilized concatemer template molecules with the soluble secondamplification primers in the presence of a primer extension polymerase,a plurality of nucleotides, and a high efficiency hybridization buffer.In some embodiment, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (e), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (f): sequencing the plurality of immobilized partially displacedforward extension strands thereby generating a first plurality ofextended reverse sequencing primer strands. In some embodiments, step(f) further comprises sequencing the plurality of immobilized detachedforward extension strands thereby generating a second plurality ofextended reverse sequencing primer strands. In some embodiments,individual immobilized partially displaced forward extension strandshave two or more extended reverse sequencing primer strands hybridizedthereon. In some embodiments, individual immobilized detached forwardextension strands have two or more extended reverse sequencing primerstrands hybridized thereon. During the sequencing of step (f), theimmobilized partially displaced forward extension strands remainhybridized to the retained immobilized concatemer template molecules.

In some embodiments, the sequencing of step (f) comprises contacting theplurality of immobilized partially displaced forward extension strands(e.g., that are hybridized to the immobilized concatemer templatemolecules), and the plurality of immobilized detached forward extensionstrands, with a plurality of soluble reverse sequencing primers under acondition suitable to hybridize the reverse sequencing primers to thereverse sequencing primer binding site of the forward extension strands(FIG. 103 ). The sequencing of step (f) comprises conducting sequencingreactions using the hybridized reverse sequencing primers wherein thereverse sequencing reactions generates a plurality of extended reversesequencing primer strands (FIG. 103 ). The extended reverse sequencingprimer strands are hybridized to a partially displaced forward extensionstrand that is hybridized to the immobilized concatemer templatemolecules, or an immobilized detached forward extension strand.

For the sake of simplicity, FIGS. 95-103 do not show an immobilizedconcatemer template molecule having a universal binding sequence for asoluble compaction oligonucleotide. The skilled artisan will appreciatethat the immobilized concatemer template molecule can include auniversal binding sequence for a soluble compaction oligonucleotide.

For the sake of simplicity, FIG. 103 shows an exemplary immobilizedpartially displaced forward extension strand that is hybridized to theimmobilized concatemer template molecule, and the immobilized detachedforward extension strand, each having one copy of an extended reversesequencing primer strand hybridized thereon. The skilled artisan willappreciate that the immobilized partially displaced forward extensionstrand that is hybridized to the immobilized concatemer templatemolecule, and the immobilized detached forward extension strand, canhave two or more copies of the extended reverse sequencing primerstrands hybridized thereon. Therefore, the reverse sequencing reactioncan generate a plurality of extended reverse sequencing primer strandshybridized to the same immobilized partially displaced forward extensionstrand that is hybridized to the immobilized concatemer templatemolecule, or the immobilized detached forward extension strand.

In some embodiments, the reverse sequencing reaction can include aplurality of compaction oligonucleotides. The compactionoligonucleotides can serve to immobilize one or more of the detachedforward extension strands via hybridization to the immobilized partiallydisplaced forward extension strand (e.g., that is hybridized to theimmobilized concatemer template molecule).

In some embodiments, in step (f), the condition that is suitable tohybridize the reverse sequencing primers to the reverse sequencingprimer binding sequences of the immobilized partially displaced forwardextension strand that is hybridized to the immobilized concatemertemplate molecule, and the immobilized detached forward extensionstrand, comprises contacting the plurality of soluble reverse sequencingprimers and the forward extension strands with a high efficiencyhybridization buffer. In some embodiments, the high efficiencyhybridization buffer comprises: (i) a first polar aprotic solvent havinga dielectric constant that is no greater than 40 and having a polarityindex of 4-9; (ii) a second polar aprotic solvent having a dielectricconstant that is no greater than 115 and is present in the hybridizationbuffer formulation in an amount effective to denature double-strandednucleic acids; (iii) a pH buffer system that maintains the pH of thehybridization buffer formulation in a range of about 4-8; and (iv) acrowding agent in an amount sufficient to enhance or facilitatemolecular crowding. In some embodiments, the high efficiencyhybridization buffer comprises: (i) the first polar aprotic solventcomprises acetonitrile at 25-50% by volume of the hybridization buffer;(ii) the second polar aprotic solvent comprises formamide at 5-10% byvolume of the hybridization buffer; (iii) the pH buffer system comprises2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) thecrowding agent comprises polyethylene glycol (PEG) at 5-35% by volume ofthe hybridization buffer. In some embodiments, the high efficiencyhybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reactions of step (f)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the immobilizedpartially displaced forward extension strand that is hybridized to theimmobilized concatemer template molecule, or the immobilized detachedforward extension strand, with one or more types of sequencingpolymerases, and a plurality of nucleotides and/or a plurality ofmultivalent molecules. In some embodiments, the soluble reversesequencing primers comprise 3′ OH extendible ends. In some embodiments,the soluble reverse sequencing primers comprise a 3′ blocking moietywhich can be removed to generate a 3′ OH extendible end. In someembodiments, the soluble reverse sequencing primers lack a nucleotidehaving a scissile moiety. The sequencing reactions that employnucleotides and/or multivalent molecules is described in more detailbelow. The reverse sequencing reactions can generate a plurality ofextended reverse sequencing primer strands. In some embodiments,individual forward extension strands have multiple copies of the reversesequencing primer binding sequences/sites, wherein each reversesequencing primer binding site is capable of hybridizing to a reversesequencing primer. Individual reverse sequencing primer binding sites ina given immobilized partially displaced forward extension strand that ishybridized to the immobilized concatemer template molecule, orimmobilized detached forward extension strand, can be hybridized to areverse sequencing primer and can undergo a sequencing reaction. Thus,an individual retained forward extension strand can undergo two or moresequence reactions, where each sequencing reaction is initiated from areverse sequencing primer that is hybridized to a reverse sequencingprimer binding site. In some embodiments, the sequencing reactionscomprise a plurality of nucleotides (or analogs thereof) labeled with adetectable reporter moiety. In some embodiments, the sequencing reactioncomprise a plurality of multivalent molecules having nucleotide units,where the multivalent molecules are labeled with a detectable reportermoiety. In some embodiments, the detectable reporter moiety comprises afluorophore.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(f). The washing step can be conducted with a washbuffer comprising a pH buffering agent, a metal chelating agent, a salt,and a detergent.

In some embodiments, the pH buffering compound in the wash buffercomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash buffer ata concentration of about 1-100 mM, or about 10-50 mM, or about 10-25 mM.In some embodiments, the pH of the pH buffering agent which is presentin any of the reagents described here in can be adjusted to a pH ofabout 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash buffercomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washbuffer comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash buffer comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash buffer can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash buffer comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash buffer at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

Introduction—Amplifying with Flexing Amplification Cycling

The present disclosure provides pairwise sequencing methods, comprisingthe general workflow: (a) providing a plurality of single strandedconcatemer template molecule comprising at least one nucleotide having ascissile moiety, the concatemer template molecules being immobilized toa first surface primer which is immobilized on the support, and thesupport comprising a plurality of immobilized first and second surfaceprimers, where the first surface primers comprise a nucleotide having ascissile moiety and the second surface primers lack a nucleotide havinga scissile moiety; (b) sequencing at least a portion of the concatemertemplate molecules and removing the extension product of the sequencingreaction; (c) hybridizing a portion of individual concatemer templatemolecules to a second surface primer, and conducting a primer extensionreaction to generate a plurality of forward extension strands that arecovalently joined to the second surface primers; (d) dissociating theimmobilized concatemer template molecules that are hybridized to thesecond surface primers and re-hybridizing a portion of individualconcatemer template molecules to a second surface primer that is notcovalently joined to a forward extension strand, by flowing onto thesupport a relaxing solution; (e) subjecting the immobilized concatemertemplate molecules and the immobilized forward extension strands totemperature dissociation and temperature re-hybridization conditions;(f) conducting an amplification reaction using the concatemer templatemolecules that are hybridized to a second surface primer to generatenewly synthesized forward extension strands each being covalently joinedto a second surface primer; (g) repeating steps (d)-(f) at least once,wherein steps (d)-(f) comprise a flexing amplification cycle; (h)generating abasic sites in the immobilized concatemer template moleculesand the immobilized first surface primers, and cleaving the abasic sitesto generate gap-containing nucleic acid molecules, thereby removing theimmobilized concatemer template molecules and the immobilized firstsurface primers from the support while retaining the immobilized forwardextension strands; and (i) sequencing the plurality of retainedimmobilized forward extension strands.

Methods for Pairwise Sequencing—Amplifying with Flexing AmplificationCycling

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a plurality of immobilized single stranded nucleicacid concatemer template molecules each comprising at least onenucleotide having a scissile moiety that can be cleaved to generate anabasic site in the concatemer template molecule, wherein individualconcatemer template molecules in the plurality are immobilized to afirst surface primer that is immobilized to a support, wherein theimmobilized first surface primers include a nucleotide having a scissilemoiety, wherein the support further comprises a plurality of immobilizedsecond surface primers which lack a nucleotide having a scissile moietyand have an extendible terminal 3′ OH group. The immobilized concatemertemplate molecules comprise two or more copies of a sequence ofinterest, two or more copies of a universal binding sequence for animmobilized first surface primer, and two or more copies of a universalbinding sequence for an immobilized second surface primer. The supportcan include an excess of immobilized first and second surface primerscompared to the number of immobilized concatemer template molecules.

In some embodiment, the immobilized concatemer template molecule canself-collapse into a compact nucleic acid nanoball. The nanoballs can beimaged and a FWHM measurement can be obtained to give the shape/size ofthe nanoballs.

In some embodiments, individual immobilized concatemer templatemolecules are covalently joined to an immobilized surface primer (e.g.,an immobilized first surface primer). In an alternative embodiment,individual immobilized concatemer template molecules are hybridized toan immobilized surface primer (e.g., an immobilized first surfaceprimer).

In some embodiments, individual concatemer template molecules in theplurality comprise two or more copies of a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of: (i) two or morecopies of a universal binding sequence for a soluble forward sequencingprimer, (ii) two or more copies of a universal binding sequence for asoluble reverse sequencing primer, (iii) two or more copies of auniversal binding sequence for an immobilized first surface primer, (iv)two or more copies of a universal binding sequence for an immobilizedsecond surface primer, (v) two or more copies of a universal bindingsequence for a first soluble amplification primer, (vi) two or morecopies of a universal binding sequence for a second solubleamplification primer, (vii) two or more copies of a universal bindingsequence for a soluble compaction oligonucleotide, (viii) two or morecopies of a sample barcode sequence and/or (ix) two or more copies of aunique molecular index sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the scissile moiety in the immobilized concatemertemplate molecules and the immobilized first surface primers of step (a)can be converted into abasic sites. In some embodiments, the scissilemoiety comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine. In the concatemer template molecules and the immobilizedfirst surface primers, the uridine can be converted to an abasic siteusing uracil DNA glycosylase (UDG). In the concatemer template moleculesand the immobilized first surface primers, the 8oxoG can be converted toan abasic site using FPG glycosylase. In the concatemer templatemolecules and the immobilized first surface primers, the deoxyinosinecan be converted to an abasic site using AlkA glycosylase.

In some embodiments, the immobilized concatemer template moleculesinclude 1-20, 20-40, 40-60, 60-80, 80-100, or a higher number ofnucleotides with a scissile moiety. In some embodiments, about 0.1-1%,or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30% or ahigher percent of the dTTP in the immobilized concatemer templatemolecules are replaced with nucleotides having a scissile moiety. Insome embodiments, the nucleotides having a scissile moiety aredistributed at random positions along individual immobilized concatemertemplate molecules. In some embodiments, the nucleotides having ascissile moiety are distributed at different positions in the differentimmobilized concatemer template molecules. In some embodiments, theimmobilized first surface primers include at least one and up to fivenucleotides having a scissile moiety.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized first surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedfirst surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized first surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a 3′ non-extendible moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the immobilized concatemer template moleculesfurther comprise two or more copies of a universal binding sequence (orcomplementary sequence thereof) for an immobilized second surface primerhaving a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized second surface primers can be immobilizedto the support or immobilized to a coating on the support. Theimmobilized second surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized second surface primers are immobilized to a supportor immobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise a moietythat blocks primer extension (e.g., non-extendible terminal 3′ end),such as for example a phosphate group, a dideoxycytidine group, aninverted dT, or an amino group. The immobilized second surface primersare not extendible in a primer extension reaction. The immobilizedsecond surface primers lack a nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (b): sequencing the plurality of immobilized concatemer templatemolecules thereby generating a plurality of extended forward sequencingprimer strands. The sequencing of step (b) comprises contacting theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers under a condition suitable tohybridize at least one forward sequencing primer to at least one of theforward sequencing primer binding sites/sequences of the immobilizedconcatemer template molecules, and conducting forward sequencingreactions using one or more types of sequencing polymerases, a pluralityof nucleotides and/or multivalent molecules, and the hybridized firstforward sequencing primers. The forward sequencing reactions cangenerate a plurality of extended forward sequencing primer strands. Insome embodiments, individual immobilized concatemer template moleculeshave multiple copies of the forward sequencing primer binding sites,wherein each forward sequencing primer binding site is capable ofhybridizing to a first forward sequencing primer. Individual forwardsequencing primer binding sites in a given immobilized concatemertemplate molecule can be hybridized to a forward sequencing primer andcan undergo a sequencing reaction. Individual immobilized concatemertemplate molecules can undergo two or more sequence reactions, whereeach sequencing reaction is initiated from a first forward sequencingprimer that is hybridized to a forward sequencing primer binding site.In some embodiments, the soluble forward sequencing primers comprise 3′OH extendible ends. In some embodiments, the soluble forward sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble forwardsequencing primers lack a nucleotide having a scissile moiety. In someembodiments, the sequencing reactions comprise a plurality ofnucleotides (or analogs thereof) labeled with a detectable reportermoiety. In some embodiments, the sequencing reaction comprise aplurality of multivalent molecules having a plurality of nucleotideunits attached to a core, where the multivalent molecules are labeledwith a detectable reporter moiety. In some embodiments, the core islabeled with a detectable reporter moiety. In some embodiments, at leastone linker and/or at least one nucleotide unit of a nucleotide arm islabeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. An exemplarynucleotide arm is shown in FIG. 108 , and exemplary multivalentmolecules are shown in FIGS. 104-107 . The sequencing reactions thatemploy nucleotides and/or multivalent molecules are described in moredetail below.

In some embodiments, the sequencing of step (b) can be conducted in thepresence or absence of a plurality of compaction oligonucleotides. Thecompaction oligonucleotides can retain the compact size and/or shape ofthe nanoballs (e.g., self-collapsed immobilized concatemer templatemolecules) during the forward sequencing reactions.

In some embodiments, the sequencing of step (b) can be conducted bycontacting the plurality of immobilized concatemer template moleculeswith a plurality of forward sequencing primers in the presence of ahybridization solution comprising pH buffering agent, a sodium salt, anda chaotropic agent. Exemplary chaotropic agents include urea, guanidinehydrochloride and guanidine thiocyanate. In some embodiments, thehybridization solution comprises MES buffering agent, NaCl and guanidinehydrochloride.

In some embodiments, the sequencing of step (b) can be conducted bycontacting the plurality of immobilized concatemer template moleculeswith a plurality of forward sequencing primers in the presence of a highefficiency hybridization buffer. In some embodiments, the highefficiency hybridization buffer comprises: (i) a first polar aproticsolvent having a dielectric constant that is no greater than 40 andhaving a polarity index of 4-9; (ii) a second polar aprotic solventhaving a dielectric constant that is no greater than 115 and is presentin the hybridization buffer formulation in an amount effective todenature double-stranded nucleic acids; (iii) a pH buffer system thatmaintains the pH of the hybridization buffer formulation in a range ofabout 4-8; and (iv) a crowding agent in an amount sufficient to enhanceor facilitate molecular crowding. In some embodiments, the highefficiency hybridization buffer comprises: (i) the first polar aproticsolvent comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) the second polar aprotic solvent comprises formamide at5-10% by volume of the hybridization buffer; (iii) the pH buffer systemcomprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5;and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35%by volume of the hybridization buffer. In some embodiments, the highefficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprisesstep (c): removing the extended forward sequencing primer strands andretaining the immobilized concatemer template molecules.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (c), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (c), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (d): generating a first plurality of immobilized forward extensionstrands by hybridizing at least one portion of individual immobilizedconcatemer template molecules to a second surface primer and conductinga primer extension reaction from the second surface primers that arehybridized to a portion of the immobilized concatemer template molecule.The primer extension reaction generates a plurality of forward extensionstrands each having a sequence that is complementary to at least aportion of the immobilized concatemer template molecules. The primerextension reaction generates a plurality of forward extension strandsthat are covalently joined to an immobilized second surface primer.

In some embodiments, the primer extension reaction of step (d) comprisesa plurality of nucleotides which lacks a nucleotide having a scissilemoiety. For example, the plurality of nucleotides comprises dATP, dGTP,dCTP and dTTP. The primer extension reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C.

The primer extension reaction of step (d) comprises a DNA polymerasecapable of catalyzing a primer extension reaction using auracil-containing template molecule (e.g., a uracil-tolerantpolymerase). Exemplary polymerases include, but are not limited to, Q5UHot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from NewEngland Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g.,mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from NewEngland Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S fromNew England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g.,catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g.,large fragment, catalog #M0275S from New England Biolabs), Bsu DNApolymerase (e.g., large fragment, catalog #M0330S from New EnglandBiolabs), Phi29 DNA polymerase (e.g., catalog #M0269S from New EnglandBiolabs), E. coli DNA polymerase (e.g., catalog #M0209S from New EnglandBiolabs), Therminator DNA polymerase (e.g., catalog #M0261S from NewEngland Biolabs), Vent DNA polymerase and Deep Vent DNA polymerase.

The primer extension reaction of step (d) comprises a polymerase havingstrand displacing activity. Examples of strand displacing polymerasesinclude phi29 DNA polymerase, large fragment of Bst DNA polymerase,large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-),Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio)

The immobilized first and second surface primers, the immobilizedconcatemer template molecules, and the immobilized forward extensionstrands, are in fluid communication with each other to permit flowingvarious solutions of buffers and reagents, and the like, onto thesupport. The immobilized first and second surface primers, theimmobilized concatemer template molecules, and the immobilized forwardextension strands can react with the solutions in a massively parallelmanner.

In some embodiments, the pairwise sequencing method further comprisesstep (e): contacting the plurality of immobilized concatemer templatemolecules and the plurality of immobilized forward extension strandswith a relaxing solution. The relaxing solution can be flowed onto thesupport to permit reaction with the immobilized concatemer templatemolecules and the immobilized forward extension strands in a massivelyparallel manner. The relaxing solution can be flowed onto the support ata temperature of about 20-25° C.

The relaxing solution comprises at least one nucleic acid relaxing agentthat can disrupt hydrogen bonding between the immobilized concatemertemplate molecules and the second surface primers. Exemplary relaxingagents include nucleic acid denaturants, chaotropic compounds, amidecompounds, aprotic compounds, primary alcohols and ethylene glycolderivatives. Chaotropic compounds comprise urea, guanidine hydrochlorideor guanidine thiocyanate. Amide compounds comprise formamide, acetamideor NN-dimethylformamide (DMF). Aprotic compounds comprise acetonitrile,DMSO (dimethyl sulfoxide), 1,4-dioxane or tetrahydrofuran. Primaryalcohols comprise 1-propanol, ethanol or methanol. Ethylene glycolderivatives comprise 1,3-propanediol, ethylene glycol, glycerol,1,2-dimethyoxyethane or 2-methoxyethanol. Other relaxing agents includesodium iodide, potassium iodide and polyamines.

The relaxing solution of step (e) can further comprise an ionic,non-ionic or zwitterion detergent. Exemplary ionic detergents includeSDS (sodium dodecyl sulfate). Exemplary non-ionic detergents includeTriton X-100, Tween 20, Tween 80 or Nonidet P-40. Exemplary zwitterionicdetergent include CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate.

The relaxing solution of step (e) can further comprise a pH bufferingcompound (e.g., zwitterionic buffering compound such as or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)).

In some embodiments, the relaxing solution of step (e) comprises any oneor a combination of two or more of a group selected from urea, guanidinehydrochloride, guanidine thiocyanate, formamide, acetamide,NN-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide),1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol,1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethyoxyethane,2-methoxyethanol, sodium iodide, potassium iodide and/or polyamines.

In some embodiments, the relaxing solution of step (e) comprisesformamide and SSC. In some embodiments, the relaxing solution comprisesacetonitrile, formamide and SSC. In some embodiments, the relaxingsolution comprises acetonitrile, formamide and MES(2-(4-morpholino)-ethane sulfonic acid). In some embodiments, therelaxing solution comprises acetonitrile, formamide, guanidinehydrochloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) and a detergent (e.g., a zwitterion detergent such as Tween-20 orTween-80). In some embodiments, the relaxing solution comprisesacetonitrile, formamide, urea and HEPES. In some embodiments, theconcentration of the SSC in the relaxing solution can be 1×, 2×, 3× or4×.

In some embodiments, the pairwise sequencing method further comprisesstep (f): dissociating the at least one portion of the immobilizedconcatemer template molecules from the immobilized second surfaceprimers and retaining the immobilized forward extension strands, andre-hybridizing at least one portion of the immobilized concatemertemplate molecules to one of the immobilized second surface primers thatare not covalently joined to a forward extension strand. In someembodiments, the nucleic acid dissociating and re-hybridizing areconducted in the presence of the relaxing solution, and comprises atemperature ramp-up, a temperature plateau, and a temperature ramp-down(e.g., FIG. 117 ). The temperature ramp-up can start at about 20-25° C.and increase to about 55-70° C. The temperature plateau can be held atabout 50-70° C. The temperature ramp-down can start at about 50-70° C.and decrease to about 20-25° C. The relaxing solution can be removedfrom the support by conducting at least one washing with a washsolution. The wash solution can include SSC (e.g., at any concentrationof about 1-5×) and a detergent (e.g., Tween-20). A skilled artisan willrecognize that the temperature ramp-up, temperature plateau, andtemperature ramp-down conditions can be modified.

In some embodiments, the forward extension strands that are duplexedwith the immobilized concatemer template molecules (e.g., generated instep (d)) can be denatured, and re-hybridized with a first surfaceprimer, in the presence of the relaxing solution, the temperatureramp-up, temperature plateau, and temperature ramp-down.

In some embodiments, the pairwise sequencing method further comprisesstep (g): contacting the re-hybridized immobilized concatemer templatemolecules with an amplification solution and conducting a primerextension reaction from the second surface primers that are rehybridizedto a portion of the immobilized concatemer template molecules togenerate a plurality of newly synthesized forward extension strandshaving a sequence that is complementary to at least a portion of theimmobilized concatemer template molecules and are covalently joined toan immobilized second surface primer. The amplifying of step (g) isconducted after the temperature ramp-up, temperature plateau,temperature ramp-down, and washing of step (f) which is described above.

In some embodiments, the forward extension strands that arere-hybridized with a first surface primer (e.g., generated in step (f)),can be contacted with the amplification solution and subjected to aprimer extension reaction to generate a plurality of newly synthesizedconcatemer template molecules when the plurality of immobilized firstsurface primers comprise a 3′ extendible end. Alternatively, when theimmobilized first surface primers comprise a 3′ non-extendible end, thenthe amplification reaction will not generate newly synthesizedconcatemer template molecules.

In some embodiments, the amplification solution of step (g) comprises aplurality of nucleotides which lacks a nucleotide having a scissilemoiety. For example, the plurality of nucleotides comprises dATP, dGTP,dCTP and dTTP. The amplification reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C.

The amplification solution of step (g) comprises a DNA polymerasecapable of catalyzing a primer extension reaction using auracil-containing template molecule (e.g., a uracil-tolerantpolymerase). Exemplary polymerases include, but are not limited to, Q5UHot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from NewEngland Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g.,mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from NewEngland Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S fromNew England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g.,catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g.,large fragment, catalog #M0275S from New England Biolabs), Bsu DNApolymerase (e.g., large fragment, catalog #M0330S from New EnglandBiolabs), Phi29 DNA polymerase (e.g., catalog #M0269S from New EnglandBiolabs), E. coli DNA polymerase (e.g., catalog #M0209S from New EnglandBiolabs), Therminator DNA polymerase (e.g., catalog #M0261S from NewEngland Biolabs), Vent DNA polymerase and Deep Vent DNA polymerase.

The amplification solution of step (g) comprises a polymerase havingstrand displacing activity. Examples of strand displacing polymerasesinclude phi29 DNA polymerase, large fragment of Bst DNA polymerase,large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-),Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

In some embodiments, the pairwise sequencing method further comprisesstep (h): conducting a flexing amplification cycle by repeating steps(e)-(g) at least once. Steps (e)-(g) can be repeated once, twice,thrice, four times, five times, six times, or up to ten times. Eachcycle can generate additional newly synthesized forward extensionstrands that are covalently joined to a second surface primer. Eachcycle can generate additional newly synthesized concatemer templatemolecules that are covalently joined to a first surface primer. Afterconducting the desired number of flexing amplification cycles, step (i)can be conducted as described directly below.

In some embodiments, the pairwise sequencing method further comprisesstep (i): removing the immobilized concatemer template molecules bygenerating abasic sites in the immobilized single stranded concatemertemplate molecules and in the immobilized first surface primers at thenucleotide(s) having the scissile moiety, and generating gaps at theabasic sites thereby generating a plurality of gap-containing nucleicacid molecules while retaining the plurality of immobilized forwardextension strands and retaining the plurality of immobilized secondsurface primers. The gap-containing nucleic acid molecules include theimmobilized concatemer template strands and the immobilized firstsurface primers.

The abasic sites are generated on the concatemer template strands andthe immobilized first surface primers that contain nucleotides havingscissile moieties. In some embodiments, the scissile moieties in theconcatemer template molecules comprises uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. The abasic sitescan be removed to generate a plurality of concatemer template moleculesand first surface primers having gaps while retaining the plurality offorward extension strands. The abasic sites can be generated by enzymethat removes the nucleo-base at the nucleotide having the scissilemoiety. The uracil in the concatemer template strands and the firstsurface primers can be converted to an abasic site using uracil DNAglycosylase (UDG). The 8oxoG in the concatemer template strands and thefirst surface primers can be converted to an abasic site using FPGglycosylase. The deoxyinosine in the concatemer template strands and thefirst surface primers can be converted to an abasic site using AlkAglycosylase.

In some embodiments, in step (i), the gaps can be generated bycontacting the abasic sites with an enzyme or a mixture of enzymeshaving lyase activity that breaks the phosphodiester backbone at the 5′and 3′ sides of the abasic site to release the base-free deoxyribose andgenerate a gap. The abasic sites can be removed using AP lyase, Endo IVendonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/AP lyase.In some embodiments, generating the abasic sites and removal of theabasic sites to generate gaps can be achieved using a mixture of uracilDNA glycosylase and DNA glycosylase-lyase endonuclease VIII, for exampleUSER (Uracil-Specific Excision Reagent Enzyme from New England Biolabs)or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (i), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical and/or heat.After the gap-removal procedure, the plurality of immobilized forwardextension strands that are covalently joined to the second surfaceprimers are retained.

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S).

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (j): sequencing the plurality of retained forward extension strandswith a plurality of soluble reverse sequencing primers therebygenerating a plurality of extended reverse sequencing primer strands. Insome embodiments, the sequencing of step (e) comprises contacting theplurality of retained forward extension strands with a plurality ofsoluble reverse sequencing primers under a condition suitable tohybridize the reverse sequencing primers to the reverse sequencingprimer binding site of the retained forward extension strands, and byconducting sequencing reactions using the hybridized reverse sequencingprimers wherein the forward sequencing reactions generates a pluralityof extended reverse sequencing primer strands.

Individual retained forward extension strands can include two or morecopies of the reverse sequencing primer strands hybridized thereon. Thereverse sequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same retained forwardextension strand.

In some embodiments, the sequencing of step (j) can be conducted in thepresence or absence of a plurality of compaction oligonucleotides. Thecompaction oligonucleotides can retain the compact size and/or shape ofthe nanoballs (e.g., self-collapsed immobilized concatemer templatemolecules) during the reverse sequencing reactions.

In some embodiments, in step (j), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a hybridization solution comprising pHbuffering agent, a sodium salt, and a chaotropic agent. Exemplarychaotropic agents include urea, guanidine hydrochloride and guanidinethiocyanate. In some embodiments, the hybridization solution comprisesMES buffering agent, NaCl and guanidine hydrochloride.

In some embodiments, in step (j), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In some embodiments, the reverse sequencing reactions of step (j)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the retainedforward extension strands, one or more types of sequencing polymerases,and a plurality of nucleotides or a plurality of multivalent molecules.In some embodiments, the soluble reverse sequencing primers comprise 3′OH extendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules are described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site. In someembodiments, the sequencing reactions comprise a plurality ofnucleotides (or analogs thereof) labeled with a detectable reportermoiety. In some embodiments, the sequencing reaction comprise aplurality of multivalent molecules having nucleotide units, where themultivalent molecules are labeled with a detectable reporter moiety. Insome embodiments, the detectable reporter moiety comprises afluorophore. The sequencing reactions that employ nucleotides and/ormultivalent molecules are described in more detail below.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(j). The washing step can be conducted with a washsolution comprising a pH buffering agent, a metal chelating agent, asalt, and a detergent.

In some embodiments, the pH buffering compound in the wash solutioncomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash solutionat a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25mM. In some embodiments, the pH of the pH buffering agent which ispresent in any of the reagents described here in can be adjusted to a pHof about 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash solutioncomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washsolution comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash solution comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash solution can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash solution comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash soli ti on at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

The wash solution can include SSC (e.g., at any concentration of about1-5×) and a detergent (e.g., Tween-20).

On Support RCA and Pairwise Sequencing—Amplifying with FlexingAmplification Cycling

The present disclosure provides pairwise sequencing methods, comprisingstep (a): providing a support having a plurality of first and secondsurface primers immobilized thereon. The first surface primers have atleast one nucleotide having a scissile moiety that can be cleaved togenerate an abasic site. The second surface primers lack a nucleotidehaving a scissile moiety and have an extendible terminal 3′OH group.

In some embodiments, the scissile moiety in the immobilized firstsurface primers of step (a) can be converted into abasic sites. In someembodiments, the scissile moiety comprises uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. In theimmobilized first surface primers, the uridine can be converted to anabasic site using uracil DNA glycosylase (UDG). In the immobilized firstsurface primers, the 8oxoG can be converted to an abasic site using FPGglycosylase. In the immobilized first surface primers, the deoxyinosinecan be converted to an abasic site using AlkA glycosylase. theimmobilized first surface primers comprise at least one and up to fivenucleotides having a scissile moiety.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized first surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedfirst surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized first surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a 3′ non-extendible moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the immobilized concatemer template moleculesfurther comprise two or more copies of a universal binding sequence (orcomplementary sequence thereof) for an immobilized second surface primerhaving a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized second surface primers can be immobilizedto the support or immobilized to a coating on the support. Theimmobilized second surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized second surface primers are immobilized to a supportor immobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise a moietythat blocks primer extension (e.g., non-extendible terminal 3′ end),such as for example a phosphate group, a dideoxycytidine group, aninverted dT, or an amino group. The immobilized second surface primersare not extendible in a primer extension reaction. The immobilizedsecond surface primers lack a nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (b): generating a plurality of immobilized single stranded nucleicacid concatemer template molecules by hybridizing a plurality ofsingle-stranded circular nucleic acid library molecules to the pluralityof immobilized first surface primers and conducting a rolling circleamplification reaction with a plurality of a strand displacingpolymerase, and a plurality of nucleotides which include dATP, dCTP,dGTP, dTTP and a plurality of nucleotides having a scissile moiety thatcan be cleaved to generate an abasic site, thereby generating aplurality of immobilized single stranded nucleic acid concatemertemplate molecules having at least one nucleotide with a scissilemoiety, wherein individual single stranded nucleic acid concatemertemplate molecules are covalently joined to an immobilized first surfaceprimer. The rolling circle amplification reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C. In some embodiments, therolling circle amplification reaction can be conducted in the presence,or in the absence, of a plurality of compaction oligonucleotides. Insome embodiments, the support comprises an excess of immobilized firstand second surface primers compared to the number of immobilizedconcatemer template molecules.

In some embodiments, the single-stranded circular nucleic acid librarymolecules comprise covalently closed circular molecules. In someembodiments, the single-stranded circular nucleic acid library moleculescan be removed from the concatemer template molecules with at least onewashing step which is conducted under a condition suitable to retain thesingle stranded nucleic acid concatemer template molecules whereindividual concatemer template molecules are operably joined to animmobilized first surface primer.

In some embodiments, each of the single stranded circular nucleic acidlibrary molecules in the plurality comprise a sequence of interest, andwherein the individual immobilized concatemer template molecules furthercomprise any one or any combination of two or more of (i) a universalbinding sequence (or complementary sequence thereof) for a solubleforward sequencing primer, (ii) a universal binding sequence (orcomplementary sequence thereof) for a soluble reverse sequencing primer,(iii) a universal binding sequence (or complementary sequence thereof)for an immobilized first surface primer, (iv) a universal bindingsequence (or complementary sequence thereof) for an immobilized secondsurface primer, (v) a universal binding sequence (or complementarysequence thereof) for a first soluble amplification primer, (vi) auniversal binding sequence (or complementary sequence thereof) for asecond soluble amplification primer, (vii) a universal binding sequence(or complementary sequence thereof) for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

In some embodiments, the rolling circle amplification reaction of step(b) generates a plurality of immobilized single stranded nucleic acidconcatemer template molecules each comprising a concatemer having atleast one nucleotide having a scissile moiety and two or more copies ofa sequence of interest, and wherein the immobilized concatemer templatemolecules further comprise any one or any combination of two or more of:(i) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a soluble forward sequencing primer,(ii) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a soluble reverse sequencing primer,(iii) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for an immobilized first surface primer,(iv) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for an immobilized second surfaceprimer, (v) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a first soluble amplificationprimer, (vi) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a second soluble amplificationprimer, (vii) two or more copies of a universal binding sequence (or acomplementary sequence thereof) for a soluble compactionoligonucleotide, (viii) two or more copies of a sample barcode sequenceand/or (ix) two or more copies of a unique molecular index sequence.

The rolling circle amplification reaction of step (b) comprises apolymerase having strand displacing activity. Examples of stranddisplacing polymerases include phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNApolymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, Deep Vent DNA polymerase and KOD DNA polymerase. Thephi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio)

The rolling circle amplification reaction of step (b) can be conductedwith a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and anucleotide having a scissile moiety to generate immobilized concatemertemplate molecules which includes at least one nucleotide having ascissile moiety. The scissile moieties in the immobilized concatemertemplate molecules can be converted into abasic sites. In someembodiments, in the nucleotide mixture, the nucleotide having thescissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine (e.g.,8oxoG) or deoxyinosine. In the immobilized concatemer templatemolecules, the uridine can be converted to an abasic site using uracilDNA glycosylase (UDG), the 8oxoG can be converted to an abasic siteusing FPG glycosylase, and the deoxyinosine can be converted to anabasic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture can include an amount ofdUTP so that a target percent of the thymidine in the resultingconcatemer molecules are replaced with dUTP. For example, when 30% ofdTTP in the concatemer molecules are to be replaced with dUTP (e.g., 30%is the target percent) then the nucleotide mixture can contain 7.5% dUTP(e.g., 30/4=7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. Thetarget percent of dTTP to be replaced by dUTP can be about 0.1-1%, orabout 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about30-45%, or about 45-50%, or a higher percent of the dTTP in theimmobilized concatemer template molecules are replaced with nucleotideshaving a scissile moiety.

In some embodiments, the nucleotide mixture can include an amount ofdeoxyinosine so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with deoxyinosine. For example, when30% of dGTP in the concatemer molecules are to be replaced withdeoxyinosine (e.g., 30% is the target percent) then the nucleotidemixture can contain 7.5% deoxyinosine (e.g., 30/4=7.5%), 17.5% dGTP, and25% each for dATP, dCTP and dTTP. The target percent of dGTP to bereplaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or about5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about45-50%, or a higher percent of the dGTP in the immobilized concatemertemplate molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the nucleotide mixture can include an amount of8oxoG so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with 8oxoG. For example, when 30% ofdGTP in the concatemer molecules are to be replaced with 8oxoG (e.g.,30% is the target percent) then the nucleotide mixture can contain 7.5%8oxoG (e.g., 30/4=7.5%), 17.5% dGTP, and 25% each for dATP, dCTP anddTTP. The target percent of dGTP to be replaced by 8oxoG can be about0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%,or about 30-45%, or about 45-50%, or a higher percent of the dGTP in theimmobilized concatemer template molecules are replaced with nucleotideshaving a scissile moiety.

In some embodiments, the rolling circle amplification reaction generatesimmobilized concatemer template molecules with incorporated nucleotideshaving a scissile moiety that are distributed at random positions alongindividual immobilized concatemer template molecules. In someembodiments, the nucleotides having a scissile moiety are distributed atdifferent positions in the different immobilized concatemer templatemolecules.

In some embodiment, the immobilized concatemer template molecule canself-collapse into a compact nucleic acid nanoball. The nanoballs can beimaged and a FWHM measurement can be obtained to give the shape/size ofthe nanoballs.

In some embodiments, the pairwise sequencing method further comprisesstep (c): sequencing the plurality of immobilized concatemer templatemolecules with a plurality of soluble forward sequencing primers therebygenerating a plurality of extended forward sequencing primer strands,wherein individual immobilized concatemer template molecules have two ormore extended forward sequencing primer strands hybridized thereon.

The sequencing of step (c) comprises contacting the plurality ofimmobilized concatemer template molecules with a plurality of solubleforward sequencing primers under a condition suitable to hybridize atleast one forward sequencing primer to at least one of the forwardsequencing primer binding sites/sequences of the immobilized concatemertemplate molecules, and conducting forward sequencing reactions usingone or more types of sequencing polymerases, a plurality of nucleotidesand/or multivalent molecules, and the hybridized first forwardsequencing primers. In some embodiments, the soluble forward sequencingprimers comprise 3′ OH extendible ends. In some embodiments, the solubleforward sequencing primers comprise a 3′ blocking moiety which can beremoved to generate a 3′ OH extendible end. In some embodiments, thesoluble forward sequencing primers lack a nucleotide having a scissilemoiety. The forward sequencing reactions can generate a plurality ofextended forward sequencing primer strands. In some embodiments,individual immobilized concatemer template molecules have multiplecopies of the forward sequencing primer binding sites, wherein eachforward sequencing primer binding site is capable of hybridizing to afirst forward sequencing primer. Individual forward sequencing primerbinding sites in a given immobilized concatemer template molecule can behybridized to a forward sequencing primer and can undergo a sequencingreaction. Individual immobilized concatemer template molecules canundergo two or more sequence reactions, where each sequencing reactionis initiated from a forward sequencing primer that is hybridized to aforward sequencing primer binding site. In some embodiments, thesequencing reactions comprise a plurality of nucleotides (or analogsthereof) labeled with a detectable reporter moiety. In some embodiments,the sequencing reaction comprise a plurality of multivalent moleculeshaving a plurality of nucleotide units attached to a core, where themultivalent molecules are labeled with a detectable reporter moiety. Insome embodiments, the core is labeled with a detectable reporter moiety.In some embodiments, at least one linker and/or at least one nucleotideunit of a nucleotide arm is labeled with a detectable reporter moiety.In some embodiments, the detectable reporter moiety comprises afluorophore. An exemplary nucleotide arm is shown in FIG. 108 , andexemplary multivalent molecules are shown in FIGS. 104-107 . Thesequencing reactions that employ nucleotides and/or multivalentmolecules are described in more detail below.

In some embodiments, the sequencing of step (c) can be conducted in thepresence or absence of a plurality of compaction oligonucleotides. Thecompaction oligonucleotides can retain the compact size and/or shape ofthe nanoballs (e.g., self-collapsed immobilized concatemer templatemolecules) during the forward sequencing reactions.

In some embodiments, the sequencing of step (c) can be conducted bycontacting the plurality of immobilized concatemer template moleculeswith a plurality of forward sequencing primers in the presence of ahybridization solution comprising pH buffering agent, a sodium salt, anda chaotropic agent. Exemplary chaotropic agents include urea, guanidinehydrochloride and guanidine thiocyanate. In some embodiments, thehybridization solution comprises MES buffering agent, NaCl and guanidinehydrochloride.

In some embodiments, the sequencing of step (c) can be conducted bycontacting the plurality of immobilized concatemer template moleculeswith a plurality of forward sequencing primers in the presence of a highefficiency hybridization buffer. In some embodiments, the highefficiency hybridization buffer comprises: (i) a first polar aproticsolvent having a dielectric constant that is no greater than 40 andhaving a polarity index of 4-9; (ii) a second polar aprotic solventhaving a dielectric constant that is no greater than 115 and is presentin the hybridization buffer formulation in an amount effective todenature double-stranded nucleic acids; (iii) a pH buffer system thatmaintains the pH of the hybridization buffer formulation in a range ofabout 4-8; and (iv) a crowding agent in an amount sufficient to enhanceor facilitate molecular crowding. In some embodiments, the highefficiency hybridization buffer comprises: (i) the first polar aproticsolvent comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) the second polar aprotic solvent comprises formamide at5-10% by volume of the hybridization buffer; (iii) the pH buffer systemcomprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5;and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35%by volume of the hybridization buffer. In some embodiments, the highefficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprisesstep (d): removing the extended forward sequencing primer strands andretaining the immobilized concatemer template molecules.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (d), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (d), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (e): generating a first plurality of immobilized forward extensionstrands by hybridizing at least one portion of individual immobilizedconcatemer template molecules to a second surface primer and conductinga primer extension reaction from the second surface primers that arehybridized to a portion of the immobilized concatemer template molecule.The primer extension reaction generates a plurality of forward extensionstrands each having a sequence that is complementary to at least aportion of the immobilized concatemer template molecules. The primerextension reaction generates a plurality of forward extension strandsthat are covalently joined to an immobilized second surface primer.

In some embodiments, the primer extension reaction of step (e) comprisesa plurality of nucleotides which lacks a nucleotide having a scissilemoiety. For example, the plurality of nucleotides comprises dATP, dGTP,dCTP and dTTP. The primer extension reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C.

The primer extension reaction of step (e) comprises a DNA polymerasecapable of catalyzing a primer extension reaction using auracil-containing template molecule (e.g., a uracil-tolerantpolymerase). Exemplary polymerases include, but are not limited to, Q5UHot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from NewEngland Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g.,mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from NewEngland Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S fromNew England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g.,catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g.,large fragment, catalog #M0275S from New England Biolabs), Bsu DNApolymerase (e.g., large fragment, catalog #M0330S from New EnglandBiolabs), Phi29 DNA polymerase (e.g., catalog #M0269S from New EnglandBiolabs), E. coli DNA polymerase (e.g., catalog #M0209S from New EnglandBiolabs), Therminator DNA polymerase (e.g., catalog #M0261S from NewEngland Biolabs), Vent DNA polymerase and Deep Vent DNA polymerase.

The primer extension reaction of step (e) comprises a polymerase havingstrand displacing activity. Examples of strand displacing polymerasesinclude phi29 DNA polymerase, large fragment of Bst DNA polymerase,large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-),Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio)

The immobilized first and second surface primers, the immobilizedconcatemer template molecules, and the immobilized forward extensionstrands, are in fluid communication with each other to permit flowingvarious solutions of buffers and reagents, and the like, onto thesupport. The immobilized first and second surface primers, theimmobilized concatemer template molecules, and the immobilized forwardextension strands can react with the solutions in a massively parallelmanner.

In some embodiments, the pairwise sequencing method further comprisesstep (f): contacting the plurality of immobilized concatemer templatemolecules and the plurality of immobilized forward extension strandswith a relaxing solution. The relaxing solution can be flowed onto thesupport to permit reaction with the immobilized concatemer templatemolecules and the immobilized forward extension strands in a massivelyparallel manner. The relaxing solution can be flowed onto the support ata temperature of about 20-25° C.

The relaxing solution comprises at least one nucleic acid relaxing agentthat can disrupt hydrogen bonding between the immobilized concatemertemplate molecules and the second surface primers. Exemplary relaxingagents include nucleic acid denaturants, chaotropic compounds, amidecompounds, aprotic compounds, primary alcohols and ethylene glycolderivatives. Chaotropic compounds comprise urea, guanidine hydrochlorideor guanidine thiocyanate. Amide compounds comprise formamide, acetamideor NN-dimethylformamide (DMF). Aprotic compounds comprise acetonitrile,DMSO (dimethyl sulfoxide), 1,4-dioxane or tetrahydrofuran. Primaryalcohols comprise 1-propanol, ethanol or methanol. Ethylene glycolderivatives comprise 1,3-propanediol, ethylene glycol, glycerol,1,2-dimethyoxyethane or 2-methoxyethanol. Other relaxing agents includesodium iodide, potassium iodide and polyamines.

The relaxing solution of step (f) can further comprise an ionic,non-ionic or zwitterion detergent. Exemplary ionic detergents includeSDS (sodium dodecyl sulfate). Exemplary non-ionic detergents includeTriton X-100, Tween 20, Tween 80 or Nonidet P-40. Exemplary zwitterionicdetergent include CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate.

The relaxing solution of step (f) can further comprise a pH bufferingcompound (e.g., zwitterionic buffering compound such as or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)).

In some embodiments, the relaxing solution of step (f) comprises any oneor a combination of two or more of a group selected from urea, guanidinehydrochloride, guanidine thiocyanate, formamide, acetamide,NN-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide),1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol,1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethyoxyethane,2-methoxyethanol, sodium iodide, potassium iodide and/or polyamines.

In some embodiments, the relaxing solution of step (f) comprisesformamide and SSC. In some embodiments, the relaxing solution comprisesacetonitrile, formamide and SSC. In some embodiments, the relaxingsolution comprises acetonitrile, formamide and MES(2-(4-morpholino)-ethane sulfonic acid). In some embodiments, therelaxing solution comprises acetonitrile, formamide, guanidinehydrochloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) and a detergent (e.g., a zwitterion detergent such as Tween-20 orTween-80). In some embodiments, the relaxing solution comprisesacetonitrile, formamide, urea and HEPES. In some embodiments, theconcentration of the SSC in the relaxing solution can be 1×, 2×, 3× or4×.

In some embodiments, the pairwise sequencing method further comprisesstep (g): dissociating the at least one portion of the immobilizedconcatemer template molecules from the immobilized second surfaceprimers and retaining the immobilized forward extension strands, andre-hybridizing at least one portion of the immobilized concatemertemplate molecules to one of the immobilized second surface primers thatare not covalently joined to a forward extension strand. In someembodiments, the nucleic acid dissociating and re-hybridizing areconducted in the presence of the relaxing solution, and comprises atemperature ramp-up, a temperature plateau, and a temperature ramp-down(e.g., FIG. 117 ). The temperature ramp-up can start at about 20-25° C.and increase to about 55-70° C. The temperature plateau can be held atabout ° C. The temperature ramp-down can start at about 50-70° C. anddecrease to about 20-25° C. The relaxing solution can be removed fromthe support by conducting at least one washing with a wash solution. Thewash solution can include SSC (e.g., at any concentration of about 1-5×)and a detergent (e.g., Tween-20). A skilled artisan will recognize thatthe temperature ramp-up, temperature plateau, and temperature ramp-downconditions can be modified.

In some embodiments, the forward extension strands that are duplexedwith the immobilized concatemer template molecules (e.g., generated instep (e)) can be denatured, and re-hybridized with a first surfaceprimer, in the presence of the relaxing solution, the temperatureramp-up, temperature plateau, and temperature ramp-down.

In some embodiments, the pairwise sequencing method further comprisesstep (h): contacting the re-hybridized immobilized concatemer templatemolecules with an amplification solution and conducting a primerextension reaction from the second surface primers that are rehybridizedto a portion of the immobilized concatemer template molecules togenerate a plurality of newly synthesized forward extension strandshaving a sequence that is complementary to at least a portion of theimmobilized concatemer template molecules and are covalently joined toan immobilized second surface primer. The amplifying of step (h) isconducted after the temperature ramp-up, temperature plateau,temperature ramp-down, and washing of step (g) which is described above.

In some embodiments, the forward extension strands that arere-hybridized with a first surface primer (e.g., generated in step (g)),can be contacted with the amplification solution and subjected to aprimer extension reaction to generate a plurality of newly synthesizedconcatemer template molecules when the plurality of immobilized firstsurface primers comprise a 3′ extendible end. Alternatively, when theimmobilized first surface primers comprise a 3′ non-extendible end, thenthe amplification reaction will not generate newly synthesizedconcatemer template molecules.

In some embodiments, the amplification solution of step (h) comprises aplurality of nucleotides which lacks a nucleotide having a scissilemoiety. For example, the plurality of nucleotides comprises dATP, dGTP,dCTP and dTTP. The amplification reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C.

The amplification solution of step (h) comprises a DNA polymerasecapable of catalyzing a primer extension reaction using auracil-containing template molecule (e.g., a uracil-tolerantpolymerase). Exemplary polymerases include, but are not limited to, Q5UHot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from NewEngland Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g.,mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from NewEngland Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S fromNew England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g.,catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g.,large fragment, catalog #M0275S from New England Biolabs), Bsu DNApolymerase (e.g., large fragment, catalog #M0330S from New EnglandBiolabs), Phi29 DNA polymerase (e.g., catalog #M0269S from New EnglandBiolabs), E. coli DNA polymerase (e.g., catalog #M0209S from New EnglandBiolabs), Therminator DNA polymerase (e.g., catalog #M0261S from NewEngland Biolabs), Vent DNA polymerase and Deep Vent DNA polymerase.

The amplification solution of step (h) comprises a polymerase havingstrand displacing activity. Examples of strand displacing polymerasesinclude phi29 DNA polymerase, large fragment of Bst DNA polymerase,large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-),Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

In some embodiments, the pairwise sequencing method further comprisesstep (i): conducting a flexing amplification cycle by repeating steps(f)-(h) at least once. Steps (f)-(h) can be repeated once, twice,thrice, four times, five times, six times, or up to ten times. Eachcycle can generate additional newly synthesized forward extensionstrands that are covalently joined to a second surface primer. Eachcycle can generate additional newly synthesized concatemer templatemolecules that are covalently joined to a first surface primer. Afterconducting the desired number of flexing amplification cycles, step (j)can be conducted as described directly below.

In some embodiments, the pairwise sequencing method further comprisesstep (j): removing the immobilized concatemer template molecules bygenerating abasic sites in the immobilized single stranded concatemertemplate molecules and in the immobilized first surface primers at thenucleotide(s) having the scissile moiety, and generating gaps at theabasic sites thereby generating a plurality of gap-containing nucleicacid molecules while retaining the plurality of immobilized forwardextension strands and retaining the plurality of immobilized secondsurface primers. The gap-containing nucleic acid molecules include theimmobilized concatemer template strands and the immobilized firstsurface primers.

The abasic sites are generated on the concatemer template strands andthe immobilized first surface primers that contain nucleotides havingscissile moieties. In some embodiments, the scissile moieties in theconcatemer template molecules comprises uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. The abasic sitescan be removed to generate a plurality of concatemer template moleculesand first surface primers having gaps while retaining the plurality offorward extension strands. The abasic sites can be generated bycontacting the immobilized concatemer template molecules and the firstsurface primers with an enzyme that removes the nucleo-base at thenucleotide having the scissile moiety. The uracil in the concatemertemplate strands and the first surface primers can be converted to anabasic site using uracil DNA glycosylase (UDG). The 8oxoG in theconcatemer template strands and the first surface primers can beconverted to an abasic site using FPG glycosylase. The deoxyinosine inthe concatemer template strands and the first surface primers can beconverted to an abasic site using AlkA glycosylase.

In some embodiments, in step (j), the gaps can be generated bycontacting the abasic sites with an enzyme or a mixture of enzymeshaving lyase activity that breaks the phosphodiester backbone at the 5′and 3′ sides of the abasic site to release the base-free deoxyribose andgenerate a gap. The abasic sites can be removed using AP lyase, Endo IVendonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/AP lyase.In some embodiments, generating the abasic sites and removal of theabasic sites to generate gaps can be achieved using a mixture of uracilDNA glycosylase and DNA glycosylase-lyase endonuclease VIII, for exampleUSER (Uracil-Specific Excision Reagent Enzyme from New England Biolabs)or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (j), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical and/or heat.After the gap-removal procedure, the plurality of immobilized forwardextension strands that are covalently joined to the second surfaceprimers are retained.

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S).

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (k): sequencing the plurality of retained forward extension strandswith a plurality of soluble reverse sequencing primers therebygenerating a plurality of extended reverse sequencing primer strands. Insome embodiments, the sequencing of step (k) comprises contacting theplurality of retained forward extension strands with a plurality ofsoluble reverse sequencing primers under a condition suitable tohybridize the reverse sequencing primers to the reverse sequencingprimer binding site of the retained forward extension strands, and byconducting sequencing reactions using the hybridized reverse sequencingprimers wherein the forward sequencing reactions generates a pluralityof extended reverse sequencing primer strands.

Individual retained forward extension strands can include two or morecopies of the reverse sequencing primer strands hybridized thereon. Thereverse sequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same retained forwardextension strand.

In some embodiments, the sequencing of step (k) can be conducted in thepresence or absence of a plurality of compaction oligonucleotides. Thecompaction oligonucleotides can retain the compact size and/or shape ofthe nanoballs (e.g., self-collapsed immobilized concatemer templatemolecules) during the reverse sequencing reactions.

In some embodiments, in step (k), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a hybridization solution comprising pHbuffering agent, a sodium salt, and a chaotropic agent. Exemplarychaotropic agents include urea, guanidine hydrochloride and guanidinethiocyanate. In some embodiments, the hybridization solution comprisesMES buffering agent, NaCl and guanidine hydrochloride.

In some embodiments, in step (k), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In some embodiments, the reverse sequencing reactions of step (k)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the retainedforward extension strands, one or more types of sequencing polymerases,and a plurality of nucleotides or a plurality of multivalent molecules.In some embodiments, the soluble reverse sequencing primers comprise 3′OH extendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules are described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site. In someembodiments, the sequencing reactions comprise a plurality ofnucleotides (or analogs thereof) labeled with a detectable reportermoiety. In some embodiments, the sequencing reaction comprise aplurality of multivalent molecules having nucleotide units, where themultivalent molecules are labeled with a detectable reporter moiety. Insome embodiments, the detectable reporter moiety comprises afluorophore. The sequencing reactions that employ nucleotides and/ormultivalent molecules are described in more detail below.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(k). The washing step can be conducted with a washsolution comprising a pH buffering agent, a metal chelating agent, asalt, and a detergent.

In some embodiments, the pH buffering compound in the wash solutioncomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash solutionat a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25mM. In some embodiments, the pH of the pH buffering agent which ispresent in any of the reagents described here in can be adjusted to a pHof about 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash solutioncomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washsolution comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash solution comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash solution can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash solution comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash soli ti on at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

The wash solution can include SSC (e.g., at any concentration of about1-5×) and a detergent (e.g., Tween-20).

In-Solution RCA and Pairwise Sequencing—Amplifying with FlexingAmplification Cycling

The present disclosure provides pairwise sequencing methods, comprisingstep (a): contacting in-solution a plurality of single-stranded circularnucleic acid library molecules to a plurality of soluble firstamplification primers, a plurality of a strand displacing polymerase,and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP anda nucleotide having a scissile moiety, under a condition suitable toform a plurality of library-primer duplexes and suitable for conductinga rolling circle amplification reaction, thereby generating a pluralityof single stranded nucleic acid concatemers having at least onenucleotide with a scissile moiety. The in-solution rolling circleamplification reaction can be conducted at an isothermal temperature ofabout 50, 51, 52, 53, 54, 55, 56, 57,58,59, 60, 61, 62, 63, 64, 65, 66,67 or 68° C.

In some embodiments, the soluble first amplification primer comprises asequence that selectively hybridizes to a universal binding sequence inthe circular nucleic acid library molecules, such as for example auniversal binding sequence (or a complementary sequence thereof) for thefirst soluble amplification primer. Alternatively, the soluble firstamplification primer comprises a random sequence that bindsnon-selectively to a sequence in the circular nucleic acid librarymolecules.

In some embodiments, individual single stranded circular nucleic acidlibrary molecules in the plurality comprises a sequence of interest andwherein the individual library molecules further comprise any one or anycombination of two or more of (i) a universal binding sequence (or acomplementary sequence thereof) for a soluble forward sequencing primer,(ii) a universal binding sequence (or a complementary sequence thereof)for a soluble reverse sequencing primer, (iii) a universal bindingsequence (or a complementary sequence thereof) for an immobilized firstsurface primer, (iv) a universal binding sequence (or a complementarysequence thereof) for an immobilized second surface primer, (v) auniversal binding sequence (or a complementary sequence thereof) for afirst soluble amplification primer, (vi) a universal binding sequence(or a complementary sequence thereof) for a second soluble amplificationprimer, (vii) a universal binding sequence (or a complementary sequencethereof) for a soluble compaction oligonucleotide, (viii) a samplebarcode sequence and/or (ix) a unique molecular index sequence. In someembodiments, the single-stranded circular nucleic acid library moleculescomprise covalently closed circular molecules.

In some embodiments, the rolling circle amplification reaction of step(a) generates a plurality of single stranded nucleic acid concatemermolecules in solution, comprising a concatemer having at least onenucleotide having a scissile moiety. In some embodiments, individualconcatemer template molecules in the plurality comprise two or morecopies of a sequence of interest, and wherein the individual immobilizedconcatemer template molecules further comprise any one or anycombination of two or more of: (i) two or more copies of a universalbinding sequence for a soluble forward sequencing primer, (ii) two ormore copies of a universal binding sequence for a soluble reversesequencing primer, (iii) two or more copies of a universal bindingsequence for an immobilized first surface primer, (iv) two or morecopies of a universal binding sequence for an immobilized second surfaceprimer, (v) two or more copies of a universal binding sequence for afirst soluble amplification primer, (vi) two or more copies of auniversal binding sequence for a second soluble amplification primer,(vii) two or more copies of a universal binding sequence for a solublecompaction oligonucleotide, (viii) two or more copies of a samplebarcode sequence and/or (ix) two or more copies of a unique molecularindex sequence.

In some embodiments, the universal binding sequence (or a complementarysequence thereof) for the forward sequencing primer can hybridize to atleast a portion of the forward sequencing primer. In some embodiments,the universal binding sequence (or a complementary sequence thereof) forthe reverse sequencing primer can hybridize to at least a portion of thereverse sequencing primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilized firstsurface primer can hybridize to at least a portion of the immobilizedfirst surface primer. In some embodiments, the universal bindingsequence (or a complementary sequence thereof) for the immobilizedsecond surface primer can hybridize to at least a portion of theimmobilized second surface primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the firstsoluble amplification primer can hybridize to at least a portion of thefirst soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the secondsoluble amplification primer can hybridize to at least a portion of thesecond soluble amplification primer. In some embodiments, the universalbinding sequence (or a complementary sequence thereof) for the solublecompaction oligonucleotide can hybridize to at least a portion of thesoluble compaction oligonucleotide.

The rolling circle amplification reaction of step (a) comprises apolymerase having strand displacing activity. Examples of stranddisplacing polymerases include phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNApolymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, Deep Vent DNA polymerase and KOD DNA polymerase. Thephi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio)

The in-solution rolling circle amplification reaction of step (a) can beconducted with a nucleotide mixture containing dATP, dCTP, dGTP, dTTPand a nucleotide having a scissile moiety to generate the concatemermolecules which includes at least one nucleotide having a scissilemoiety. The scissile moieties in the concatemer molecules can beconverted into abasic sites. In some embodiments, in the nucleotidemixture, the nucleotide having the scissile moiety comprises uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. In theconcatemer molecules, the uridine can be converted to an abasic siteusing uracil DNA glycosylase (UDG), the 8oxoG can be converted to anabasic site using FPG glycosylase, and the deoxyinosine can be convertedto an abasic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture can include an amount ofdUTP so that a target percent of the thymidine in the resultingconcatemer molecules are replaced with dUTP. For example, when 30% ofdTTP in the concatemer molecules are to be replaced with dUTP (e.g., 30%is the target percent) then the nucleotide mixture can contain 7.5% dUTP(e.g., 30/4=7.5%), 17.5% dTTP, and 25% each for dATP, dCTP and dGTP. Thetarget percent of dTTP to be replaced by dUTP can be about 0.1-1%, orabout 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about30-45%, or about 45-50%, or a higher percent of the dTTP in theconcatemer molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the nucleotide mixture can include an amount ofdeoxyinosine so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with deoxyinosine. For example, when30% of dGTP in the concatemer molecules are to be replaced withdeoxyinosine (e.g., 30% is the target percent) then the nucleotidemixture can contain 7.5% deoxyinosine (e.g., 30/4=7.5%), 17.5% dGTP, and25% each for dATP, dCTP and dTTP. The target percent of dGTP to bereplaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or about5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about45-50%, or a higher percent of the dGTP in the concatemer molecules arereplaced with nucleotides having a scissile moiety.

In some embodiments, the nucleotide mixture can include an amount of8oxoG so that a target percent of the guanosine in the resultingconcatemer molecules are replaced with 8oxoG. For example, when 30% ofdGTP in the concatemer molecules are to be replaced with 8oxoG (e.g.,30% is the target percent) then the nucleotide mixture can contain 7.5%8oxoG (e.g., 30/4=7.5%), 17.5% dGTP, and 25% each for dATP, dCTP anddTTP. The target percent of dGTP to be replaced by 8oxoG can be about0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%,or about 30-45%, or about 45-50%, or a higher percent of the dGTP in theconcatemer molecules are replaced with nucleotides having a scissilemoiety.

In some embodiments, the in-solution rolling circle amplificationreaction generates concatemer molecules with incorporated nucleotideshaving a scissile moiety that are distributed at random positions alongindividual immobilized concatemer template molecules. In someembodiments, the nucleotides having a scissile moiety are distributed atdifferent positions in the different concatemer molecules.

In some embodiments, the pairwise sequencing method further comprisesstep (b): distributing the rolling circle amplification reaction fromstep (a) onto a support having a plurality of the first and secondsurface primers immobilized thereon, under a condition suitable forhybridizing one or more portions of individual single strandedconcatemers to one or more immobilized first surface primers. In someembodiments, the immobilized first surface primers include at least onenucleotide having a scissile moiety. In some embodiments, theimmobilized second surface primers lack a nucleotide having a scissilemoiety and have an extendible 3′OH group.

In some embodiments, the distributing of step (b) can be conducted inthe presence of a hybridization solution comprising pH buffering agent,a sodium salt, and a chaotropic agent. Exemplary chaotropic agentsinclude urea, guanidine hydrochloride and guanidine thiocyanate. In someembodiments, the hybridization solution comprises MES buffering agent,NaCl and guanidine hydrochloride.

In some embodiments, the distributing of step (b) can be conducted inthe presence of a high efficiency hybridization buffer. In someembodiments, the high efficiency hybridization buffer comprises: (i) afirst polar aprotic solvent having a dielectric constant that is nogreater than 40 and having a polarity index of 4-9; (ii) a second polaraprotic solvent having a dielectric constant that is no greater than 115and is present in the hybridization buffer formulation in an amounteffective to denature double-stranded nucleic acids; (iii) a pH buffersystem that maintains the pH of the hybridization buffer formulation ina range of about 4-8; and (iv) a crowding agent in an amount sufficientto enhance or facilitate molecular crowding. In some embodiments, thehigh efficiency hybridization buffer comprises: (i) the first polaraprotic solvent comprises acetonitrile at 25-50% by volume of thehybridization buffer; (ii) the second polar aprotic solvent comprisesformamide at 5-10% by volume of the hybridization buffer; (iii) the pHbuffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at apH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol(PEG) at 5-35% by volume of the hybridization buffer. In someembodiments, the high efficiency hybridization buffer further comprisesbetaine.

In some embodiments, the pairwise sequencing method further comprisesstep (c): continuing the rolling circle amplification reaction on thesupport to generate a plurality of extended concatemer templatemolecules that are immobilized via hybridization to the immobilizedfirst surface primers. The on-support RCA reaction can be conducted witha plurality of a strand displacing polymerase, and a plurality ofnucleotides which include dATP, dCTP, dGTP, dTTP and a nucleotide havinga scissile moiety, under a condition suitable to generate a plurality ofextended concatemers having at least one nucleotide with a scissilemoiety. In some embodiments, the rolling circle amplification reactionon the support can be conducted in the presence, or in the absence, of aplurality of compaction oligonucleotides. The rolling circleamplification reaction on the support can be conducted at an isothermaltemperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59, 60, 61, 62,63, 64, 65, 66, 67 or 68° C.

The rolling circle amplification reaction of step (c) continues on thesupport using a polymerase having strand displacing activity. Examplesof strand displacing polymerases include phi29 DNA polymerase, largefragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase(exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNApolymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase.The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g.,MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., fromThermo Fisher Scientific), or chimeric QualiPhi DNA polymerase (e.g.,from 4basebio)

In some embodiments, the on-support rolling circle amplificationreaction generates immobilized concatemer template molecules withincorporated nucleotides having a scissile moiety that are distributedat random positions along individual immobilized concatemer templatemolecules. In some embodiments, the nucleotides having a scissile moietyare distributed at different positions in the different immobilizedconcatemer template molecules.

In some embodiments, the support comprises an excess of immobilizedfirst and second surface primers compared to the number of immobilizedconcatemer template molecules.

In some embodiment, the immobilized concatemer template molecule canself-collapse into a compact nucleic acid nanoball. The nanoballs can beimaged and a FWHM measurement can be obtained to give the shape/size ofthe nanoballs.

In some embodiments, the immobilized first surface primers comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized first surface primers can be immobilized tothe support or immobilized to a coating on the support. The immobilizedfirst surface primers can be embedded and attached (coupled) to thecoating on the support. In some embodiments, the 5′ end of theimmobilized first surface primers are immobilized to a support orimmobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized first surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized first surface primershaving the same sequence. The immobilized first surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths. In some embodiments, the 3′terminal end of the immobilized first surface primers comprise anextendible 3′ OH moiety. In some embodiments, the 3′ terminal end of theimmobilized first surface primers comprise a 3′ non-extendible moiety.

In some embodiments, the plurality of immobilized first surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the first surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized firstsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their 5′ ends. In some embodiments, the plurality ofimmobilized first surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the first surface primers resistant to exonucleasedegradation.

In some embodiments, the immobilized first surface primers comprise atleast one locked nucleic acid (LNA) which comprises a methylene bridgebond between a 2′ oxygen and 4′ carbon of the pentose ring. Immobilizedfirst surface primers that include at least one LNA can be resistant tonuclease digestions and can exhibit increased melting temperature whenhybridized to the forward extension strand.

In some embodiments, the immobilized concatemer template moleculesfurther comprise two or more copies of a universal binding sequence (orcomplementary sequence thereof) for an immobilized second surface primerhaving a sequence that differs from the first immobilized surfaceprimer. The immobilized second surface primers of step (a) comprisesingle stranded oligonucleotides comprising DNA, RNA or a combination ofDNA and RNA. The immobilized second surface primers can be immobilizedto the support or immobilized to a coating on the support. Theimmobilized second surface primers can be embedded and attached(coupled) to the coating on the support. In some embodiments, the 5′ endof the immobilized second surface primers are immobilized to a supportor immobilized to a coating on the support. Alternatively, an interiorportion or the 3′ end of the immobilized second surface primers can beimmobilized to a support or immobilized to a coating on the support. Thesupport comprises a plurality of immobilized second surface primershaving the same sequence. The immobilized second surface primers can beany length, for example 4-50 nucleotides, or 50-100 nucleotides, or100-150 nucleotides, or longer lengths.

In some embodiments, the 3′ terminal end of the immobilized secondsurface primers comprise an extendible 3′ OH moiety. In someembodiments, the 3′ terminal end of the immobilized second surfaceprimers comprise a 3′ non-extendible moiety. In some embodiments, the 3′terminal end of the immobilized second surface primers comprise a moietythat blocks primer extension (e.g., non-extendible terminal 3′ end),such as for example a phosphate group, a dideoxycytidine group, aninverted dT, or an amino group. The immobilized second surface primersare not extendible in a primer extension reaction. The immobilizedsecond surface primers lack a nucleotide having a scissile moiety.

In some embodiments, the plurality of immobilized second surface primerscomprise at least one phosphorothioate diester bond at their 5′ endswhich can render the second surface primers resistant to exonucleasedegradation. In some embodiments, the plurality of immobilized secondsurface primers comprise 2-5 or more consecutive phosphorothioatediester bonds at their ends. In some embodiments, the plurality ofimmobilized second surface primers comprise at least one ribonucleotideand/or at least one 2′-O-methyl or 2′-O-methoxyethyl (MOE) nucleotidewhich can render the second surface primers resistant to exonucleasedegradation.

In some embodiments, the support comprises about 10²-10¹⁵ immobilizedfirst surface primers per mm². In some embodiments, the supportcomprises about 10²-10¹⁵ immobilized second surface primers per mm². Insome embodiments, the support comprises about 10²-10¹⁵ immobilized firstsurface primers and immobilized second surface primers per mm².

The immobilized surface primers (e.g., first and second surface primers)are in fluid communication with each other to permit flowing varioussolutions of linear or circular nucleic acid template molecules, solubleprimers, enzymes, nucleotides, divalent cations, buffers, reagents, andthe like, onto the support so that the plurality of immobilized surfaceprimers (and the primer extension products generated from theimmobilized surface primers) react with the solutions in a massivelyparallel manner.

In some embodiments, the pairwise sequencing method further comprisesstep (d): sequencing the plurality of immobilized concatemer templatemolecules with a plurality of soluble forward sequencing primers therebygenerating a plurality of extended forward sequencing primer strands,wherein individual immobilized concatemer template molecules have two ormore extended forward sequencing primer strands hybridized thereon.

The sequencing of step (d) comprises contacting the plurality ofimmobilized concatemer template molecules with a plurality of solubleforward sequencing primers under a condition suitable to hybridize atleast one forward sequencing primer to at least one of the forwardsequencing primer binding sites/sequences of the immobilized concatemertemplate molecules, and conducting forward sequencing reactions usingone or more types of sequencing polymerases, a plurality of nucleotidesand/or multivalent molecules, and the hybridized first forwardsequencing primers. In some embodiments, the soluble forward sequencingprimers comprise 3′ OH extendible ends. In some embodiments, the solubleforward sequencing primers comprise a 3′ blocking moiety which can beremoved to generate a 3′ OH extendible end. In some embodiments, thesoluble forward sequencing primers lack a nucleotide having a scissilemoiety. The forward sequencing reactions can generate a plurality ofextended forward sequencing primer strands. In some embodiments,individual immobilized concatemer template molecules have multiplecopies of the forward sequencing primer binding sites, wherein eachforward sequencing primer binding site is capable of hybridizing to afirst forward sequencing primer. Individual forward sequencing primerbinding sites in a given immobilized concatemer template molecule can behybridized to a forward sequencing primer and can undergo a sequencingreaction. Individual immobilized concatemer template molecules canundergo two or more sequence reactions, where each sequencing reactionis initiated from a forward sequencing primer that is hybridized to aforward sequencing primer binding site. In some embodiments, thesequencing reactions comprise a plurality of nucleotides (or analogsthereof) labeled with a detectable reporter moiety. In some embodiments,the sequencing reaction comprise a plurality of multivalent moleculeshaving a plurality of nucleotide units attached to a core, where themultivalent molecules are labeled with a detectable reporter moiety. Insome embodiments, the core is labeled with a detectable reporter moiety.In some embodiments, at least one linker and/or at least one nucleotideunit of a nucleotide arm is labeled with a detectable reporter moiety.In some embodiments, the detectable reporter moiety comprises afluorophore. An exemplary nucleotide arm is shown in FIG. 108 , andexemplary multivalent molecules are shown in FIGS. 104-107 . Thesequencing reactions that employ nucleotides and/or multivalentmolecules are described in more detail below.

In some embodiments, the sequencing of step (d) can be conducted in thepresence or absence of a plurality of compaction oligonucleotides. Thecompaction oligonucleotides can retain the compact size and/or shape ofthe nanoballs (e.g., self-collapsed immobilized concatemer templatemolecules) during the forward sequencing reactions.

In some embodiments, the sequencing of step (d) can be conducted bycontacting the plurality of immobilized concatemer template moleculeswith a plurality of forward sequencing primers in the presence of ahybridization solution comprising pH buffering agent, a sodium salt, anda chaotropic agent. Exemplary chaotropic agents include urea, guanidinehydrochloride and guanidine thiocyanate. In some embodiments, thehybridization solution comprises MES buffering agent, NaCl and guanidinehydrochloride.

In some embodiments, the sequencing of step (d) can be conducted bycontacting the plurality of immobilized concatemer template moleculeswith a plurality of forward sequencing primers in the presence of a highefficiency hybridization buffer. In some embodiments, the highefficiency hybridization buffer comprises: (i) a first polar aproticsolvent having a dielectric constant that is no greater than 40 andhaving a polarity index of 4-9; (ii) a second polar aprotic solventhaving a dielectric constant that is no greater than 115 and is presentin the hybridization buffer formulation in an amount effective todenature double-stranded nucleic acids; (iii) a pH buffer system thatmaintains the pH of the hybridization buffer formulation in a range ofabout 4-8; and (iv) a crowding agent in an amount sufficient to enhanceor facilitate molecular crowding. In some embodiments, the highefficiency hybridization buffer comprises: (i) the first polar aproticsolvent comprises acetonitrile at 25-50% by volume of the hybridizationbuffer; (ii) the second polar aprotic solvent comprises formamide at5-10% by volume of the hybridization buffer; (iii) the pH buffer systemcomprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5;and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35%by volume of the hybridization buffer. In some embodiments, the highefficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprisesstep (e): removing the extended forward sequencing primer strands andretaining the immobilized concatemer template molecules.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using an enzyme or a chemicalreagent. For example, the plurality of extended forward sequencingprimer strands can be enzymatically degraded using a 5′ to 3′double-stranded DNA exonuclease, including T7 exonuclease (e.g., fromNew England Biolabs, catalog #M0263S). In some embodiments, theplurality of extended forward sequencing primer strands can be removedwith a temperature that favors nucleic acid denaturation.

In some embodiments, in step (e), a denaturation reagent can be used toremove the plurality of extended forward sequencing primer strands,wherein the denaturation reagent comprises any one or any combination ofcompounds such as formamide, acetonitrile, guanidinium chloride and/or apH buffering agent (e.g., Tris-HCl, MES, HEPES, MOPS, or the like).Optionally, the denaturation reagent can further comprise PEG.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using an elevated temperature(e.g., heat) with or without a nucleic acid denaturation reagent. Theplurality of extended forward sequencing primer strands can be subjectedto a temperature of about 45-50° C., or about 50-60° C., or about 60-70°C., or about 70-80° C., or about 80-90° C., or about 90-95° C., orhigher temperature.

In some embodiments, in step (e), the plurality of extended forwardsequencing primer strands can be removed using 100% formamide at atemperature of about 65° C. for about 3 minutes, and washing with areagent comprising about 50 mM NaCl or equivalent ionic strength andhaving a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (f): generating a first plurality of immobilized forward extensionstrands by hybridizing at least one portion of individual immobilizedconcatemer template molecules to a second surface primer and conductinga primer extension reaction from the second surface primers that arehybridized to a portion of the immobilized concatemer template molecule.The primer extension reaction generates a plurality of forward extensionstrands each having a sequence that is complementary to at least aportion of the immobilized concatemer template molecules. The primerextension reaction generates a plurality of forward extension strandsthat are covalently joined to an immobilized second surface primer.

In some embodiments, the primer extension reaction of step (f) comprisesa plurality of nucleotides which lacks a nucleotide having a scissilemoiety. For example, the plurality of nucleotides comprises dATP, dGTP,dCTP and dTTP. The primer extension reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C.

The primer extension reaction of step (f) comprises a DNA polymerasecapable of catalyzing a primer extension reaction using auracil-containing template molecule (e.g., a uracil-tolerantpolymerase). Exemplary polymerases include, but are not limited to, Q5UHot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from NewEngland Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g.,mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from NewEngland Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S fromNew England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g.,catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g.,large fragment, catalog #M0275S from New England Biolabs), Bsu DNApolymerase (e.g., large fragment, catalog #M0330S from New EnglandBiolabs), Phi29 DNA polymerase (e.g., catalog #M0269S from New EnglandBiolabs), E. coli DNA polymerase (e.g., catalog #M0209S from New EnglandBiolabs), Therminator DNA polymerase (e.g., catalog #M0261S from NewEngland Biolabs), Vent DNA polymerase and Deep Vent DNA polymerase.

The primer extension reaction of step (f) comprises a polymerase havingstrand displacing activity. Examples of strand displacing polymerasesinclude phi29 DNA polymerase, large fragment of Bst DNA polymerase,large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-),Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio)

The immobilized first and second surface primers, the immobilizedconcatemer template molecules, and the immobilized forward extensionstrands, are in fluid communication with each other to permit flowingvarious solutions of buffers and reagents, and the like, onto thesupport. The immobilized first and second surface primers, theimmobilized concatemer template molecules, and the immobilized forwardextension strands can react with the solutions in a massively parallelmanner.

In some embodiments, the pairwise sequencing method further comprisesstep (g): contacting the plurality of immobilized concatemer templatemolecules and the plurality of immobilized forward extension strandswith a relaxing solution. The relaxing solution can be flowed onto thesupport to permit reaction with the immobilized concatemer templatemolecules and the immobilized forward extension strands in a massivelyparallel manner. The relaxing solution can be flowed onto the support ata temperature of about 20-25° C.

The relaxing solution comprises at least one nucleic acid relaxing agentthat can disrupt hydrogen bonding between the immobilized concatemertemplate molecules and the second surface primers. Exemplary relaxingagents include nucleic acid denaturants, chaotropic compounds, amidecompounds, aprotic compounds, primary alcohols and ethylene glycolderivatives. Chaotropic compounds comprise urea, guanidine hydrochlorideor guanidine thiocyanate. Amide compounds comprise formamide, acetamideor NN-dimethylformamide (DMF). Aprotic compounds comprise acetonitrile,DMSO (dimethyl sulfoxide), 1,4-dioxane or tetrahydrofuran. Primaryalcohols comprise 1-propanol, ethanol or methanol. Ethylene glycolderivatives comprise 1,3-propanediol, ethylene glycol, glycerol,1,2-dimethyoxyethane or 2-methoxyethanol. Other relaxing agents includesodium iodide, potassium iodide and polyamines.

The relaxing solution of step (g) can further comprise an ionic,non-ionic or zwitterion detergent. Exemplary ionic detergents includeSDS (sodium dodecyl sulfate). Exemplary non-ionic detergents includeTriton X-100, Tween 20, Tween 80 or Nonidet P-40. Exemplary zwitterionicdetergent include CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate.

The relaxing solution of step (g) can further comprise a pH bufferingcompound (e.g., zwitterionic buffering compound such as or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)).

In some embodiments, the relaxing solution of step (g) comprises any oneor a combination of two or more of a group selected from urea, guanidinehydrochloride, guanidine thiocyanate, formamide, acetamide,NN-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide),1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol,1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethyoxyethane,2-methoxyethanol, sodium iodide, potassium iodide and/or polyamines.

In some embodiments, the relaxing solution of step (g) comprisesformamide and SSC. In some embodiments, the relaxing solution comprisesacetonitrile, formamide and SSC. In some embodiments, the relaxingsolution comprises acetonitrile, formamide and MES(2-(4-morpholino)-ethane sulfonic acid). In some embodiments, therelaxing solution comprises acetonitrile, formamide, guanidinehydrochloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) and a detergent (e.g., a zwitterion detergent such as Tween-20 orTween-80). In some embodiments, the relaxing solution comprisesacetonitrile, formamide, urea and HEPES. In some embodiments, theconcentration of the SSC in the relaxing solution can be 1×, 2×, 3× or4×.

In some embodiments, the pairwise sequencing method further comprisesstep (h): dissociating the at least one portion of the immobilizedconcatemer template molecules from the immobilized second surfaceprimers and retaining the immobilized forward extension strands, andre-hybridizing at least one portion of the immobilized concatemertemplate molecules to one of the immobilized second surface primers thatare not covalently joined to a forward extension strand. In someembodiments, the nucleic acid dissociating and re-hybridizing areconducted in the presence of the relaxing solution, and comprises atemperature ramp-up, a temperature plateau, and a temperature ramp-down(e.g., FIG. 117 ). The temperature ramp-up can start at about 20-25° C.and increase to about 55-70° C. The temperature plateau can be held atabout 50-70° C. The temperature ramp-down can start at about 50-70° C.and decrease to about 20-25° C. The relaxing solution can be removedfrom the support by conducting at least one washing with a washsolution. The wash solution can include SSC (e.g., at any concentrationof about 1-5×) and a detergent (e.g., Tween-20). A skilled artisan willrecognize that the temperature ramp-up, temperature plateau, andtemperature ramp-down conditions can be modified.

In some embodiments, the forward extension strands that are duplexedwith the immobilized concatemer template molecules (e.g., generated instep (f)) can be denatured, and re-hybridized with a first surfaceprimer, in the presence of the relaxing solution, the temperatureramp-up, temperature plateau, and temperature ramp-down.

In some embodiments, the pairwise sequencing method further comprisesstep (i): contacting the re-hybridized immobilized concatemer templatemolecules with an amplification solution and conducting a primerextension reaction from the second surface primers that arere-hybridized to a portion of the immobilized concatemer templatemolecules to generate a plurality of newly synthesized forward extensionstrands having a sequence that is complementary to at least a portion ofthe immobilized concatemer template molecules and are covalently joinedto an immobilized second surface primer. The amplifying of step (i) isconducted after the temperature ramp-up, temperature plateau,temperature ramp-down, and washing of step (h) which is described above.

In some embodiments, the forward extension strands that arere-hybridized with a first surface primer (e.g., generated in step (h)),can be contacted with the amplification solution and subjected to aprimer extension reaction to generate a plurality of newly synthesizedconcatemer template molecules when the plurality of immobilized firstsurface primers comprise a 3′ extendible end. Alternatively, when theimmobilized first surface primers comprise a 3′ non-extendible end, thenthe amplification reaction will not generate newly synthesizedconcatemer template molecules.

In some embodiments, the amplification solution of step (i) comprises aplurality of nucleotides which lacks a nucleotide having a scissilemoiety. For example, the plurality of nucleotides comprises dATP, dGTP,dCTP and dTTP. The amplification reaction can be conducted at anisothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57,58,59,60, 61, 62, 63, 64, 65, 66, 67 or 68° C.

The amplification solution of step (i) comprises a DNA polymerasecapable of catalyzing a primer extension reaction using auracil-containing template molecule (e.g., a uracil-tolerantpolymerase). Exemplary polymerases include, but are not limited to, Q5UHot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from NewEngland Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g.,mixture of Taq and Deep Vent DNA polymerases, catalog #M0480S from NewEngland Biolabs), LongAmp Taq DNA polymerase (e.g., catalog #M0323S fromNew England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g.,catalog #M0490S from New England Biolabs), Bst DNA polymerase (e.g.,large fragment, catalog #M0275S from New England Biolabs), Bsu DNApolymerase (e.g., large fragment, catalog #M0330S from New EnglandBiolabs), Phi29 DNA polymerase (e.g., catalog #M0269S from New EnglandBiolabs), E. coli DNA polymerase (e.g., catalog #M0209S from New EnglandBiolabs), Therminator DNA polymerase (e.g., catalog #M0261S from NewEngland Biolabs), Vent DNA polymerase and Deep Vent DNA polymerase.

The amplification solution of step (i) comprises a polymerase havingstrand displacing activity. Examples of strand displacing polymerasesinclude phi29 DNA polymerase, large fragment of Bst DNA polymerase,large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-),Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reversetranscriptase, HIV viral reverse transcriptase, Deep Vent DNA polymeraseand KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

In some embodiments, the pairwise sequencing method further comprisesstep (j): conducting a flexing amplification cycle by repeating steps(g)-(i) at least once. Steps (g)-(i) can be repeated once, twice,thrice, four times, five times, six times, or up to ten times. Eachcycle can generate additional newly synthesized forward extensionstrands that are covalently joined to a second surface primer. Eachcycle can generate additional newly synthesized concatemer templatemolecules that are covalently joined to a first surface primer. Afterconducting the desired number of flexing amplification cycles, step (k)can be conducted as described directly below.

In some embodiments, the pairwise sequencing method further comprisesstep (k): removing the immobilized concatemer template molecules bygenerating abasic sites in the immobilized single stranded concatemertemplate molecules and in the immobilized first surface primers at thenucleotide(s) having the scissile moiety, and generating gaps at theabasic sites thereby generating a plurality of gap-containing nucleicacid molecules while retaining the plurality of immobilized forwardextension strands and retaining the plurality of immobilized secondsurface primers. The gap-containing nucleic acid molecules include theimmobilized concatemer template strands and the immobilized firstsurface primers.

The abasic sites are generated on the concatemer template strands andthe immobilized first surface primers that contain nucleotides havingscissile moieties. In some embodiments, the scissile moieties in theconcatemer template molecules comprises uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. The abasic sitescan be removed to generate a plurality of concatemer template moleculesand first surface primers having gaps while retaining the plurality offorward extension strands. The abasic sites can be generated bycontacting the immobilized concatemer template molecules and the firstsurface primers with an enzyme that removes the nucleo-base at thenucleotide having the scissile moiety. The uracil in the concatemertemplate strands and the first surface primers can be converted to anabasic site using uracil DNA glycosylase (UDG). The 8oxoG in theconcatemer template strands and the first surface primers can beconverted to an abasic site using FPG glycosylase. The deoxyinosine inthe concatemer template strands and the first surface primers can beconverted to an abasic site using AlkA glycosylase.

In some embodiments, in step (k), the gaps can be generated bycontacting the abasic sites with an enzyme or a mixture of enzymeshaving lyase activity that breaks the phosphodiester backbone at the 5′and 3′ sides of the abasic site to release the base-free deoxyribose andgenerate a gap. The abasic sites can be removed using AP lyase, Endo IVendonuclease, FPG glycosylase/AP lyase, Endo VIII glycosylase/AP lyase.In some embodiments, generating the abasic sites and removal of theabasic sites to generate gaps can be achieved using a mixture of uracilDNA glycosylase and DNA glycosylase-lyase endonuclease VIII, for exampleUSER (Uracil-Specific Excision Reagent Enzyme from New England Biolabs)or thermolabile USER (also from New England Biolabs).

In some embodiments, in step (k), the plurality of gap-containingtemplate molecules can be removed using an enzyme, chemical and/or heat.After the gap-removal procedure, the plurality of immobilized forwardextension strands that are covalently joined to the second surfaceprimers are retained.

For example, the plurality of gap-containing template molecules can beenzymatically degraded using a 5′ to 3′ double-stranded DNA exonuclease,including T7 exonuclease (e.g., from New England Biolabs, catalog#M0263S).

In some embodiments, the plurality of gap-containing template moleculescan be removed using a chemical reagent that favors nucleic aciddenaturation. The denaturation reagent can include any one or anycombination of compounds such as formamide, acetonitrile, guanidiniumchloride and/or a buffering agent (e.g., Tris-HCl, MES, HEPES, or thelike).

In some embodiments, the plurality of gap-containing template moleculescan be removed using an elevated temperature (e.g., heat) with orwithout a nucleic acid denaturation reagent. The gap-containing templatemolecules can be subjected to a temperature of about 45-50° C., or about50-60° C., or about 60-70° C., or about 70-80° C., or about 80-90° C.,or about 90-95° C., or higher temperature.

In some embodiments, the plurality of gap-containing template moleculescan be removed using 100% formamide at a temperature of about 65° C. forabout 3 minutes, and washing with a reagent comprising about 50 mM NaClor equivalent ionic strength and having a pH of about 6.5-8.5.

In some embodiments, the pairwise sequencing method further comprisesstep (l): sequencing the plurality of retained forward extension strandswith a plurality of soluble reverse sequencing primers therebygenerating a plurality of extended reverse sequencing primer strands. Insome embodiments, the sequencing of step (l) comprises contacting theplurality of retained forward extension strands with a plurality ofsoluble reverse sequencing primers under a condition suitable tohybridize the reverse sequencing primers to the reverse sequencingprimer binding site of the retained forward extension strands, and byconducting sequencing reactions using the hybridized reverse sequencingprimers wherein the forward sequencing reactions generates a pluralityof extended reverse sequencing primer strands.

Individual retained forward extension strands can include two or morecopies of the reverse sequencing primer strands hybridized thereon. Thereverse sequencing reaction can generate a plurality of extended reversesequencing primer strands hybridized to the same retained forwardextension strand.

In some embodiments, the sequencing of step (l) can be conducted in thepresence or absence of a plurality of compaction oligonucleotides. Thecompaction oligonucleotides can retain the compact size and/or shape ofthe nanoballs (e.g., self-collapsed immobilized concatemer templatemolecules) during the reverse sequencing reactions.

In some embodiments, in step (l), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a hybridization solution comprising pHbuffering agent, a sodium salt, and a chaotropic agent. Exemplarychaotropic agents include urea, guanidine hydrochloride and guanidinethiocyanate. In some embodiments, the hybridization solution comprisesMES buffering agent, NaCl and guanidine hydrochloride.

In some embodiments, in step (l), the condition suitable to hybridizethe reverse sequencing primers to the reverse sequencing primer bindingsequences of the retained forward extension strands comprises contactingthe plurality of soluble reverse sequencing primers and the retainedforward extension strands with a high efficiency hybridization buffer.In some embodiments, the high efficiency hybridization buffer comprises:(i) a first polar aprotic solvent having a dielectric constant that isno greater than 40 and having a polarity index of 4-9; (ii) a secondpolar aprotic solvent having a dielectric constant that is no greaterthan 115 and is present in the hybridization buffer formulation in anamount effective to denature double-stranded nucleic acids; (iii) a pHbuffer system that maintains the pH of the hybridization bufferformulation in a range of about 4-8; and (iv) a crowding agent in anamount sufficient to enhance or facilitate molecular crowding. In someembodiments, the high efficiency hybridization buffer comprises: (i) thefirst polar aprotic solvent comprises acetonitrile at 25-50% by volumeof the hybridization buffer; (ii) the second polar aprotic solventcomprises formamide at 5-10% by volume of the hybridization buffer;(iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid(MES) at a pH of 5-6.5; and (iv) the crowding agent comprisespolyethylene glycol (PEG) at 5-35% by volume of the hybridizationbuffer. In some embodiments, the high efficiency hybridization bufferfurther comprises betaine.

In some embodiments, the reverse sequencing reactions of step (1)comprises contacting the plurality of soluble reverse sequencing primerswith the reverse sequencing primer binding sequences of the retainedforward extension strands, one or more types of sequencing polymerases,and a plurality of nucleotides or a plurality of multivalent molecules.In some embodiments, the soluble reverse sequencing primers comprise 3′OH extendible ends. In some embodiments, the soluble reverse sequencingprimers comprise a 3′ blocking moiety which can be removed to generate a3′ OH extendible end. In some embodiments, the soluble reversesequencing primers lack a nucleotide having a scissile moiety. Thesequencing reactions that employ nucleotides and/or multivalentmolecules are described in more detail below. The reverse sequencingreactions can generate a plurality of extended reverse sequencing primerstrands. In some embodiments, individual retained forward extensionstrands have multiple copies of the reverse sequencing primer bindingsequences/sites, wherein each reverse sequencing primer binding site iscapable of hybridizing to a reverse sequencing primer. Individualreverse sequencing primer binding sites in a given retained forwardextension strand can be hybridized to a reverse sequencing primer andcan undergo a sequencing reaction. Thus, an individual retained forwardextension strand can undergo two or more sequence reactions, where eachsequencing reaction is initiated from a reverse sequencing primer thatis hybridized to a reverse sequencing primer binding site. In someembodiments, the sequencing reactions comprise a plurality ofnucleotides (or analogs thereof) labeled with a detectable reportermoiety. In some embodiments, the sequencing reaction comprise aplurality of multivalent molecules having nucleotide units, where themultivalent molecules are labeled with a detectable reporter moiety. Insome embodiments, the detectable reporter moiety comprises afluorophore. The sequencing reactions that employ nucleotides and/ormultivalent molecules are described in more detail below.

In some embodiments, at least one washing step can be conducted afterany of steps (a)-(l). The washing step can be conducted with a washsolution comprising a pH buffering agent, a metal chelating agent, asalt, and a detergent.

In some embodiments, the pH buffering compound in the wash solutioncomprises any one or any combination of two or more of Tris, Tris-HCl,Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES,CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (a.k.a 2-amino methanol;MEA), a citrate compound, a citrate mixture, NaOH and/or KOH. In someembodiments, the pH buffering agent can be present in the wash solutionat a concentration of about 1-100 mM, or about 10-50 mM, or about 10-25mM. In some embodiments, the pH of the pH buffering agent which ispresent in any of the reagents described here in can be adjusted to a pHof about 4-9, or a pH of about 5-9, or a pH of about 5-8.

In some embodiments, the metal chelating agent in the wash solutioncomprises EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycoltetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid),DPTA (diethylene triamine pentaacetic acid), NTA(N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate,calcium citrate, ammonium citrate, ammonium bicitrate, citric acid,potassium citrate, or magnesium citrate. In some embodiments, the washsolution comprises a chelating agent at a concentration of about 0.01-50mM, or about 0.1-20 mM, or about 0.2-10 mM.

In some embodiments, the salt in the wash solution comprises NaCl, KCl,NH₂SO₄ or potassium glutamate. In some embodiments, the detergentcomprises an ionic detergent such as SDS (sodium dodecyl sulfate). Thewash solution can include a monovalent salt at a concentration of about25-500 mM, or about 50-250 mM, or about 100-200 mM.

In some embodiments, the detergent in the wash solution comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (lithium dodecyl sulfate),sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate,sodium deoxycholate or sodium cholate. In some embodiments, thedetergent is included in the wash soli ti on at a concentration of about0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%,or about 0.2-0.25%.

The wash solution can include SSC (e.g., at any concentration of about1-5×) and a detergent (e.g., Tween-20).

Supports and Low Non-Specific Coatings

The present disclosure provides pairwise sequencing compositions andmethods which employ a support comprising a plurality of oligonucleotidesurface primers immobilized thereon. In some embodiments, the support ispassivated with a low non-specific binding coating. The surface coatingsdescribed herein exhibit very low non-specific binding to reagentstypically used for nucleic acid capture, amplification and sequencingworkflows, such as dyes, nucleotides, enzymes, and nucleic acid primers.The surface coatings exhibit low background fluorescence signals or highcontrast-to-noise (CNR) ratios compared to conventional surfacecoatings.

The low non-specific binding coating comprises one layer or multiplelayers (FIG. 115 ). In some embodiments, the plurality of surfaceprimers are immobilized to the low non-specific binding coating. In someembodiments, at least one surface primer is embedded within the lownon-specific binding coating. The low non-specific binding coatingenables improved nucleic acid hybridization and amplificationperformance. In general, the supports comprise a substrate (or supportstructure), one or more layers of a covalently or non-covalentlyattached low-binding, chemical modification layers, e.g., silane layers,polymer films, and one or more covalently or non-covalently attachedsurface primers that can be used for tethering single-stranded nucleicacid library molecules to the support. In some embodiments, theformulation of the coating, e.g., the chemical composition of one ormore layers, the coupling chemistry used to cross-link the one or morelayers to the support and/or to each other, and the total number oflayers, may be varied such that non-specific binding of proteins,nucleic acid molecules, and other hybridization and amplificationreaction components to the coating is minimized or reduced relative to acomparable monolayer. The formulation of the coating described hereinmay be varied such that non-specific hybridization on the coating isminimized or reduced relative to a comparable monolayer. The formulationof the coating may be varied such that non-specific amplification on thecoating is minimized or reduced relative to a comparable monolayer. Theformulation of the coating may be varied such that specificamplification rates and/or yields on the coating are maximized.Amplification levels suitable for detection are achieved in no more than2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30amplification cycles in some cases disclosed herein.

The support structure that comprises the one or more chemically-modifiedlayers, e.g., layers of a low non-specific binding polymer, may beindependent or integrated into another structure or assembly. Forexample, in some embodiments, the support structure may comprise one ormore surfaces within an integrated or assembled microfluidic flow cell.The support structure may comprise one or more surfaces within amicroplate format, e.g., the bottom surface of the wells in amicroplate. In some embodiments, the support structure comprises theinterior surface (such as the lumen surface) of a capillary. In someembodiments, the support structure comprises the interior surface (suchas the lumen surface) of a capillary etched into a planar chip.

The attachment chemistry used to graft a first chemically-modified layerto the surface of the support will generally be dependent on both thematerial from which the surface is fabricated and the chemical nature ofthe layer. In some embodiments, the first layer may be covalentlyattached to the surface. In some embodiments, the first layer may benon-covalently attached, e.g., adsorbed to the support throughnon-covalent interactions such as electrostatic interactions, hydrogenbonding, or van der Waals interactions between the support and themolecular components of the first layer. In either case, the support maybe treated prior to attachment or deposition of the first layer. Any ofa variety of surface preparation techniques known to those of skill inthe art may be used to clean or treat the surface. For example, glass orsilicon surfaces may be acid-washed using a Piranha solution (a mixtureof sulfuric acid (H₂SO₄) and hydrogen peroxide (H₂O₂)), base treatmentin KOH and NaOH, and/or cleaned using an oxygen plasma treatment method.

Silane chemistries constitute non-limiting approaches for covalentlymodifying the silanol groups on glass or silicon surfaces to attach morereactive functional groups (e.g., amines or carboxyl groups), which maythen be used in coupling linker molecules (e.g., linear hydrocarbonmolecules of various lengths, such as C6, C12, C18 hydrocarbons, orlinear polyethylene glycol (PEG) molecules) or layer molecules (e.g.,branched PEG molecules or other polymers) to the surface. Examples ofsuitable silanes that may be used in creating any of the disclosed lowbinding coatings include, but are not limited to, (3-Aminopropyl)trimethoxysilane (APTMS), (3-Aminopropyl) triethoxysilane (APTES), anyof a variety of PEG-silanes (e.g., comprising molecular weights of 1K,2K, 5K, 10K, 20K, etc.), amino-PEG silane (i.e., comprising a free aminofunctional group), maleimide-PEG silane, biotin-PEG silane, and thelike.

Any of a variety of molecules known to those of skill in the artincluding, but not limited to, amino acids, peptides, nucleotides,oligonucleotides, other monomers or polymers, or combinations thereofmay be used in creating the one or more chemically-modified layers onthe support, where the choice of components used may be varied to alterone or more properties of the layers, e.g., the surface density offunctional groups and/or tethered oligonucleotide primers, thehydrophilicity/hydrophobicity of the layers, or the threethree-dimensional nature (i.e., “thickness”) of the layer. Examples ofpolymers that may be used to create one or more layers of lownon-specific binding material in any of the disclosed coatings include,but are not limited to, polyethylene glycol (PEG) of various molecularweights and branching structures, streptavidin, polyacrylamide,polyester, dextran, poly-lysine, and poly-lysine copolymers, or anycombination thereof. Examples of conjugation chemistries that may beused to graft one or more layers of material (e.g. polymer layers) tothe surface and/or to cross-link the layers to each other include, butare not limited to, biotin-streptavidin interactions (or variationsthereof), his tag—Ni/NTA conjugation chemistries, methoxy etherconjugation chemistries, carboxylate conjugation chemistries, amineconjugation chemistries, NHS esters, maleimides, thiol, epoxy, azide,hydrazide, alkyne, isocyanate, and silane.

The low non-specific binding surface coating may be applied uniformlyacross the support. Alternatively, the surface coating may be patterned,such that the chemical modification layers are confined to one or morediscrete regions of the support. For example, the coating may bepatterned using photolithographic techniques to create an ordered arrayor random pattern of chemically-modified regions on the support.Alternately or in combination, the coating may be patterned using, e.g.,contact printing and/or ink-jet printing techniques. In someembodiments, an ordered array or random pattern of chemically-modifiedregions may comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions.

In some embodiments, the low nonspecific binding coatings comprisehydrophilic polymers that are non-specifically adsorbed or covalentlygrafted to the support. Typically, passivation is performed utilizingpoly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) orpolyoxyethylene) or other hydrophilic polymers with different molecularweights and end groups that are linked to a support using, for example,silane chemistry. The end groups distal from the surface can include,but are not limited to, biotin, methoxy ether, carboxylate, amine, NHSester, maleimide, and bis-silane. In some embodiments, two or morelayers of a hydrophilic polymer, e.g., a linear polymer, branchedpolymer, or multi-branched polymer, may be deposited on the surface. Insome embodiments, two or more layers may be covalently coupled to eachother or internally cross-linked to improve the stability of theresulting coating. In some embodiments, surface primers with differentnucleotide sequences and/or base modifications (or other biomolecules,e.g., enzymes or antibodies) may be tethered to the resulting layer atvarious surface densities. In some embodiments, for example, bothsurface functional group density and surface primer concentration may bevaried to attain a desired surface primer density range. Additionally,surface primer density can be controlled by diluting the surface primerswith other molecules that carry the same functional group. For example,amine-labeled surface primers can be diluted with amine-labeledpolyethylene glycol in a reaction with an NETS-ester coated surface toreduce the final primer density. Surface primers with different lengthsof linker between the hybridization region and the surface attachmentfunctional group can also be applied to control surface density. Exampleof suitable linkers include poly-T and poly-A strands at the 5′ end ofthe primer (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20 monomerunits), and carbon-chain (e.g., C6, C12, C18, etc.). To measure theprimer density, fluorescently-labeled primers may be tethered to thesurface and a fluorescence reading then compared with that for a dyesolution of known concentration.

In some embodiments, the low nonspecific binding coatings comprise afunctionalized polymer coating layer covalently bound at least to aportion of the support via a chemical group on the support, a primergrafted to the functionalized polymer coating, and a water-solubleprotective coating on the primer and the functionalized polymer coating.In some embodiments, the functionalized polymer coating comprises apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM).

In order to scale primer surface density and add additionaldimensionality to hydrophilic or amphoteric coatings, supportscomprising multi-layer coatings of PEG and other hydrophilic polymershave been developed. By using hydrophilic and amphoteric surfacelayering approaches that include, but are not limited to, thepolymer/co-polymer materials described below, it is possible to increaseprimer loading density on the support significantly. Traditional PEGcoating approaches use monolayer primer deposition, which have beengenerally reported for single molecule applications, but do not yieldhigh copy numbers for nucleic acid amplification applications. Asdescribed herein “layering” can be accomplished using traditionalcrosslinking approaches with any compatible polymer or monomer subunitssuch that a surface comprising two or more highly crosslinked layers canbe built sequentially. Examples of suitable polymers include, but arenot limited to, streptavidin, poly acrylamide, polyester, dextran,poly-lysine, and copolymers of poly-lysine and PEG. In some embodiments,the different layers may be attached to each other through any of avariety of conjugation reactions including, but not limited to,biotin-streptavidin binding, azide-alkyne click reaction, amine-NETSester reaction, thiol-maleimide reaction, and ionic interactions betweenpositively charged polymer and negatively charged polymer. In someembodiments, high primer density materials may be constructed insolution and subsequently layered onto the surface in multiple steps.

Examples of materials from which the support structure may be fabricatedinclude, but are not limited to, glass, fused-silica, silicon, a polymer(e.g., polystyrene (PS), macroporous polystyrene (MPPS),polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP),polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET)), or any combination thereof. Various compositionsof both glass and plastic support structures are contemplated.

The support structure may be rendered in any of a variety of geometriesand dimensions known to those of skill in the art, and may comprise anyof a variety of materials known to those of skill in the art. Forexample, the support structure may be locally planar (e.g., comprising amicroscope slide or the surface of a microscope slide). Globally, thesupport structure may be cylindrical (e.g., comprising a capillary orthe interior surface of a capillary), spherical (e.g., comprising theouter surface of a non-porous bead), or irregular (e.g., comprising theouter surface of an irregularly-shaped, non-porous bead or particle). Insome embodiments, the surface of the support structure used for nucleicacid hybridization and amplification may be a solid, non-porous surface.In some embodiments, the surface of the support structure used fornucleic acid hybridization and amplification may be porous, such thatthe coatings described herein penetrate the porous surface, and nucleicacid hybridization and amplification reactions performed thereon mayoccur within the pores.

The support structure that comprises the one or more chemically-modifiedlayers, e.g., layers of a low non-specific binding polymer, may beindependent or integrated into another structure or assembly. Forexample, the support structure may comprise one or more surfaces withinan integrated or assembled microfluidic flow cell. The support structuremay comprise one or more surfaces within a microplate format, e.g., thebottom surface of the wells in a microplate. In some embodiments, thesupport structure comprises the interior surface (such as the lumensurface) of a capillary. In some embodiments the support structurecomprises the interior surface (such as the lumen surface) of acapillary etched into a planar chip.

As noted, the low non-specific binding supports of the presentdisclosure exhibit reduced non-specific binding of proteins, nucleicacids, and other components of the hybridization and/or amplificationformulation used for solid-phase nucleic acid amplification. The degreeof non-specific binding exhibited by a given support surface may beassessed either qualitatively or quantitatively. For example, exposureof the surface to fluorescent dyes (e.g., cyanins such as Cy3, or Cy5,etc., fluoresceins, coumarins, rhodamines, etc. or other dyes disclosedherein), fluorescently-labeled nucleotides, fluorescently-labeledoligonucleotides, and/or fluorescently-labeled proteins (e.g.polymerases) under a standardized set of conditions, followed by aspecified rinse protocol and fluorescence imaging may be used as aqualitative tool for comparison of non-specific binding on supportscomprising different surface formulations. In some embodiments, exposureof the surface to fluorescent dyes, fluorescently-labeled nucleotides,fluorescently-labeled oligonucleotides, and/or fluorescently-labeledproteins (e.g. polymerases) under a standardized set of conditions,followed by a specified rinse protocol and fluorescence imaging may beused as a quantitative tool for comparison of non-specific binding onsupports comprising different surface formulations—provided that carehas been taken to ensure that the fluorescence imaging is performedunder conditions where fluorescence signal is linearly related (orrelated in a predictable manner) to the number of fluorophores on thesupport surface (e.g., under conditions where signal saturation and/orself-quenching of the fluorophore is not an issue) and suitablecalibration standards are used. In some embodiments, other techniquesknown to those of skill in the art, for example, radioisotope labelingand counting methods may be used for quantitative assessment of thedegree to which non-specific binding is exhibited by the differentsupport surface formulations of the present disclosure.

Some surfaces disclosed herein exhibit a ratio of specific tononspecific binding of a fluorophore such as Cy3 of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,50, 75, 100, or greater than 100, or any intermediate value spanned bythe range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to nonspecific fluorescence of a fluorophore such as Cy3 of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or anyintermediate value spanned by the range herein.

The degree of non-specific binding exhibited by the disclosedlow-binding supports may be assessed using a standardized protocol forcontacting the surface with a labeled protein (e.g., bovine serumalbumin (BSA), streptavidin, a DNA polymerase, a reverse transcriptase,a helicase, a single-stranded binding protein (SSB), etc., or anycombination thereof), a labeled nucleotide, a labeled oligonucleotide,etc., under a standardized set of incubation and rinse conditions,followed be detection of the amount of label remaining on the surfaceand comparison of the signal resulting therefrom to an appropriatecalibration standard. In some embodiments, the label may comprise afluorescent label. In some embodiments, the label may comprise aradioisotope. In some embodiments, the label may comprise any otherdetectable label known to one of skill in the art. In some embodiments,the degree of non-specific binding exhibited by a given support surfaceformulation may thus be assessed in terms of the number ofnon-specifically bound protein molecules (or nucleic acid molecules orother molecules) per unit area. In some embodiments, the low-bindingsupports of the present disclosure may exhibit non-specific proteinbinding (or non-specific binding of other specified molecules, (e.g.,cyanins such as Cy3, or Cy5, etc., fluoresceins, coumarins, rhodamines,etc. or other dyes disclosed herein)) of less than 0.001 molecule perμm², less than 0.01 molecule per μm², less than 0.1 molecule per μm²,less than 0.25 molecule per μm², less than 0.5 molecule per μm², lessthan 1 molecule per μm², less than 10 molecules per μm², less than 100molecules per μm², or less than 1,000 molecules per μm². Those of skillin the art will realize that a given support surface of the presentdisclosure may exhibit non-specific binding falling anywhere within thisrange, for example, of less than 86 molecules per μm². For example, somemodified surfaces disclosed herein exhibit nonspecific protein bindingof less than 0.5 molecule/μm² following contact with a 1 μM solution ofCy3 labeled streptavidin (GE Amersham) in phosphate buffered saline(PBS) buffer for 15 minutes, followed by 3 rinses with deionized water.Some modified surfaces disclosed herein exhibit nonspecific binding ofCy3 dye molecules of less than 0.25 molecules per μm². In independentnonspecific binding assays, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5SA dye (ThermoFisher), 10 μM Aminoallyl-dUTP-ATTO-647N (JenaBiosciences), 10 μM Aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10μM Aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM7-Propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences, and 10 μM7-Propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated onthe low binding coated supports at 37° C. for 15 minutes in a 384 wellplate format. Each well was rinsed 2-3× with 50 ul deionized RNase/DNaseFree water and 2-3× with 25 mM ACES buffer pH 7.4. The 384 well plateswere imaged on a GE Typhoon instrument using the Cy3, AF555, or Cy5filter sets (according to dye test performed) as specified by themanufacturer at a PMT gain setting of 800 and resolution of 50-100 μm.For higher resolution imaging, images were collected on an Olympus IX83microscope (e.g., inverted fluorescence microscope) (Olympus Corp.,Center Valley, Pa.) with a total internal reflectance fluorescence(TIRF) objective (100×, 1.5 NA, Olympus), a CCD camera (e.g., an OlympusEM-CCD monochrome camera, Olympus XM-10 monochrome camera, or an OlympusDP80 color and monochrome camera), an illumination source (e.g., anOlympus 100 W Hg lamp, an Olympus 75 W Xe lamp, or an Olympus U-HGLGPSfluorescence light source), and excitation wavelengths of 532 nm or 635nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science,LLC, Rochester, N.Y.), e.g., 405, 488, 532, or 633 nm dichroicreflectors/beamsplitters, and band pass filters were chosen as 532 LP or645 LP concordant with the appropriate excitation wavelength. Somemodified surfaces disclosed herein exhibit nonspecific binding of dyemolecules of less than 0.25 molecules per μm². In some embodiments, thecoated support was immersed in a buffer (e.g., 25 mM ACES, pH 7.4) whilethe image was acquired.

In some embodiments, the surfaces disclosed herein exhibit a ratio ofspecific to nonspecific binding of a fluorophore such as Cy3 of at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate valuespanned by the range herein. In some embodiments, the surfaces disclosedherein exhibit a ratio of specific to nonspecific fluorescence signalsfor a fluorophore such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, orgreater than 100, or any intermediate value spanned by the range herein.

The low-background surfaces consistent with the disclosure herein mayexhibit specific dye attachment (e.g., Cy3 attachment) to non-specificdye adsorption (e.g., Cy3 dye adsorption) ratios of at least 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50specific dye molecules attached per molecule nonspecifically adsorbed.Similarly, when subjected to an excitation energy, low-backgroundsurfaces consistent with the disclosure herein to which fluorophores,e.g., Cy3, have been attached may exhibit ratios of specificfluorescence signal (e.g., arising from Cy3-labeled oligonucleotidesattached to the surface) to non-specific adsorbed dye fluorescencesignals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1,30:1, 40:1, 50:1, or more than 50:1.

In some embodiments, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed support surfaces may be assessed,for example, through the measurement of water contact angles in which asmall droplet of water is placed on the surface and its angle of contactwith the surface is measured using, e.g., an optical tensiometer. Insome embodiments, a static contact angle may be determined. In someembodiments, an advancing or receding contact angle may be determined.In some embodiments, the water contact angle for the hydrophilic,low-binding support surfaced disclosed herein may range from about 0degrees to about 30 degrees. In some embodiments, the water contactangle for the hydrophilic, low-binding support surfaced disclosed hereinmay no more than 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases thecontact angle is no more than 40 degrees. Those of skill in the art willrealize that a given hydrophilic, low-binding support surface of thepresent disclosure may exhibit a water contact angle having a value ofanywhere within this range.

In some embodiments, the hydrophilic surfaces disclosed hereinfacilitate reduced wash times for bioassays, often due to reducednonspecific binding of biomolecules to the low-binding surfaces. In someembodiments, adequate wash steps may be performed in less than 60, 50,40, 30, 20, 15, 10, or less than 10 seconds. For example, adequate washsteps may be performed in less than 30 seconds.

Some low-binding surfaces of the present disclosure exhibit significantimprovement in stability or durability to prolonged exposure to solventsand elevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. For example, the stability of the disclosedsurfaces may be tested by fluorescently labeling a functional group onthe surface, or a tethered biomolecule (e.g., an oligonucleotide primer)on the surface, and monitoring fluorescence signal before, during, andafter prolonged exposure to solvents and elevated temperatures, or torepeated cycles of solvent exposure or changes in temperature. In someembodiments, the degree of change in the fluorescence used to assess thequality of the surface may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, or 25% over a time period of 1 minute, 2 minutes, 3 minutes, 4minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposureto solvents and/or elevated temperatures (or any combination of thesepercentages as measured over these time periods). In some embodiments,the degree of change in the fluorescence used to assess the quality ofthe surface may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25%over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900cycles, or 1,000 cycles of repeated exposure to solvent changes and/orchanges in temperature (or any combination of these percentages asmeasured over this range of cycles).

In some embodiments, the surfaces disclosed herein may exhibit a highratio of specific signal to nonspecific signal or other background. Forexample, when used for nucleic acid amplification, some surfaces mayexhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greater than asignal of an adjacent unpopulated region of the surface. Similarly, somesurfaces exhibit an amplification signal that is at least 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greaterthan a signal of an adjacent amplified nucleic acid population region ofthe surface.

In some embodiments, fluorescence images of the disclosed low backgroundsurfaces when used in nucleic acid hybridization or amplificationapplications to create polonies of hybridized or clonally-amplifiednucleic acid molecules (e.g., that have been directly or indirectlylabeled with a fluorophore) exhibit contrast-to-noise ratios (CNRs) ofat least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than250.

One or more types of primer may be attached or tethered to the supportsurface. In some embodiments, the one or more types of adapters orprimers may comprise spacer sequences, adapter sequences forhybridization to adapter-ligated target library nucleic acid sequences,forward amplification primers, reverse amplification primers, sequencingprimers, and/or molecular barcoding sequences, or any combinationthereof. In some embodiments, 1 primer or adapter sequence may betethered to at least one layer of the surface. In some embodiments, atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer oradapter sequences may be tethered to at least one layer of the surface.

In some embodiments, the tethered adapter and/or primer sequences mayrange in length from about 10 nucleotides to about 100 nucleotides. Insome embodiments, the tethered adapter and/or primer sequences may be atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, or at least 100 nucleotidesin length. In some embodiments, the tethered adapter and/or primersequences may be at most 100, at most 90, at most 80, at most 70, atmost 60, at most 50, at most 40, at most 30, at most 20, or at most 10nucleotides in length. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some embodiments the length of thetethered adapter and/or primer sequences may range from about 20nucleotides to about 80 nucleotides. Those of skill in the art willrecognize that the length of the tethered adapter and/or primersequences may have any value within this range, e.g., about 24nucleotides.

In some embodiments, the resultant surface density of primers (e.g.,capture primers) on the low binding support surfaces of the presentdisclosure may range from about 100 primer molecules per μm² to about100,000 primer molecules per μm². In some embodiments, the resultantsurface density of primers on the low binding support surfaces of thepresent disclosure may range from about 1,000 primer molecules per μm²to about 1,000,000 primer molecules per μm². In some embodiments, thesurface density of primers may be at least 1,000, at least 10,000, atleast 100,000, or at least 1,000,000 molecules per μm². In someembodiments, the surface density of primers may be at most 1,000,000, atmost 100,000, at most 10,000, or at most 1,000 molecules per μm². Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome embodiments the surface density of primers may range from about10,000 molecules per μm² to about 100,000 molecules per μm². Those ofskill in the art will recognize that the surface density of primermolecules may have any value within this range, e.g., about 455,000molecules per μm². In some embodiments, the surface density of targetlibrary nucleic acid sequences initially hybridized to adapter or primersequences on the support surface may be less than or equal to thatindicated for the surface density of tethered primers. In someembodiments, the surface density of clonally-amplified target librarynucleic acid sequences hybridized to adapter or primer sequences on thesupport surface may span the same range as that indicated for thesurface density of tethered primers.

Local densities as listed above do not preclude variation in densityacross a surface, such that a surface may comprise a region having anoligo density of, for example, 500,000/μm², while also comprising atleast a second region having a substantially different local density.

In some embodiments, the performance of nucleic acid hybridizationand/or amplification reactions using the disclosed reaction formulationsand low-binding supports may be assessed using fluorescence imagingtechniques, where the contrast-to-noise ratio (CNR) of the imagesprovides a key metric in assessing amplification specificity andnon-specific binding on the support. CNR is described in U.S. patentapplication US 2020/0149095, hereby expressly incorporated by referencein its entirety. CNR is commonly defined as:CNR=(Signal-Background)/Noise. The background term is commonly taken tobe the signal measured for the interstitial regions surrounding aparticular feature (diffraction limited spot, DLS) in a specified regionof interest (ROI). While signal-to-noise ratio (SNR) is often consideredto be a benchmark of overall signal quality, it can be shown thatimproved CNR can provide a significant advantage over SNR as a benchmarkfor signal quality in applications that require rapid image capture(e.g., sequencing applications for which cycle times must be minimized),as shown in the example below. At high CNR the imaging time required toreach accurate discrimination (and thus accurate base-calling in thecase of sequencing applications) can be drastically reduced even withmoderate improvements in CNR. Improved CNR in imaging data on theimaging integration time provides a method for more accurately detectingfeatures such as clonally-amplified nucleic acid colonies on the supportsurface.

In most ensemble-based sequencing approaches, the background term istypically measured as the signal associated with ‘interstitial’ regions.In addition to “interstitial” background (B_(inter) ), “intrastitial”background (B_(intra) ) exists within the region occupied by anamplified DNA colony. The combination of these two background signalsdictates the achievable CNR, and subsequently directly impacts theoptical instrument requirements, architecture costs, reagent costs,run-times, cost/genome, and ultimately the accuracy and data quality forcyclic array-based sequencing applications. The B_(inter) backgroundsignal arises from a variety of sources; a few examples includeauto-fluorescence from consumable flow cells, non-specific adsorption ofdetection molecules that yield spurious fluorescence signals that mayobscure the signal from the ROI, the presence of non-specific DNAamplification products (e.g., those arising from primer dimers). Intypical next generation sequencing (NGS) applications, this backgroundsignal in the current field-of-view (FOV) is averaged over time andsubtracted. The signal arising from individual DNA colonies (i.e.,(Signal)-B(interstial) in the FOV) yields a discernable feature that canbe classified. In some embodiments, the intrastitial background(B(intrastitial)) can contribute a confounding fluorescence signal thatis not specific to the target of interest, but is present in the sameROI thus making it far more difficult to average and subtract.

Nucleic acid amplification on the low-binding coated supports describedherein may decrease the B(interstitial) background signal by reducingnon-specific binding, may lead to improvements in specific nucleic acidamplification, and may lead to a decrease in non-specific amplificationthat can impact the background signal arising from both the interstitialand intrastitial regions. In some embodiments, the disclosed low-bindingcoated supports, optionally used in combination with the disclosedhybridization and/or amplification reaction formulations, may lead toimprovements in CNR by a factor of 2, 5, 10, 100, 250, 500 or 1000-foldover those achieved using conventional supports and hybridization,amplification, and/or sequencing protocols. Although described here inthe context of using fluorescence imaging as the read-out or detectionmode, the same principles apply to the use of the disclosed low-bindingcoated supports and nucleic acid hybridization and amplificationformulations for other detection modes as well, including both opticaland non-optical detection modes.

Compositions and method for preparing, testing and using the lownon-specific binding coatings, including methods for immobilizing to thecoating any type of primer (e.g., capture oligonucleotides,amplification oligonucleotides and/or circularization oligonucleotides)are described in U.S. Pat. No. 10,768,173, issued Sep. 8, 2020; U.S.application Ser. No. 16/363,842, filed Mar. 25, 2019; Ser. No.16/740,355, filed Jan. 10, 2020; Ser. No. 16/740,357, filed Jan. 10,2020; and U.S. Pat. No. 16,855,877, filed Apr. 22, 2020. The contents ofthe aforementioned issued patent and patent applications are herebyexpressly incorporated by reference in their entireties.

Library Molecules

The pairwise sequencing compositions and methods described herein employnucleic acid library molecules which typically refers to a population ofnucleic acid molecules each comprising a sequence of interest (e.g.,insert region) covalently joined to at least one adaptor sequence.Individual library molecules in the population can have an insertsequence that is the same or different insert sequence as other librarymolecules in the population. Individual library molecules in thepopulation can have an adaptor sequence that is the same (e.g., auniversal adaptor sequence) or different (e.g., unique identificationsequence) adaptor sequence as other library molecules in the population.

The nucleic acid library molecule comprises DNA, RNA, cDNA or chimericDNA/RNA. The nucleic acid library molecule can be single-stranded ordouble-stranded, or can include single-stranded or double-strandedportions. The nucleic acid library molecule can be linear, covalentlyclosed circular, dumbbell, hairpin or other forms.

The insert region of a nucleic acid library molecule comprises asequence of interest extracted from any source including a biologicalsample (e.g., fresh or live sample) such as a single cell, a pluralityof cells or tissue. The insert region can be isolated from healthy ordiseases cells or tissues. The insert region can be obtained from anarchived sample such as a fresh frozen paraffin embedded (FFPE) sample,or from needle biopsies, circulating tumor cells, cell free circulatingDNA. Cells or tissues are typically treated with a lysis buffer torelease their DNA and RNA, and the desired nucleic acid is separatedfrom non-desired macromolecules such as proteins.

The insert region of a nucleic acid library molecule can be isolated inany form, including chromosomal, genomic (e.g., whole genomic),organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinantmolecules, cloned or amplified.

The insert region can be prepared using recombinant nucleic acidtechnology including but not limited to any combination of vectorcloning, transgenic host cell preparation, host cell culturing and/orPCR amplification.

The insert region can be prepared using chemical synthesis proceduresusing native nucleotides with or without nucleotide analogs or modifiednucleotide linkages that confer certain properties, including resistanceto enzymatic digestion, or increased thermal stability. Examples ofnucleotide analogs and modified nucleotide linkages that inhibitnuclease digestion include phosphorothioate, 2′-O-methyl RNA, inverteddT, and 2′3′ dideoxy-dT. Insert regions that include locked nucleicacids (LNA) have increased thermal stability.

The insert region can be isolated from any organism including viruses,fungi, prokaryotes or eukaryotes. The insert region can be isolated fromany organism including human, simian, ape, canine, feline, bovine,equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insector bacteria. The insert region can be isolated from organisms borne inair, water, soil or food.

The insert region can be isolated from any biological fluid, includingblood, urine, serum, lymph, tumor, saliva, anal secretions, vaginalsecretions, amniotic samples, perspiration, semen, environmental samplesor culture samples. The insert region can be isolated from any organ,including head, neck, brain, breast, ovary, cervix, colon, rectum,endometrium, gallbladder, intestines, bladder, prostate, testicles,liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus,skin, heart, larynx, or other organs.

The insert region can be in fragmented or un-fragmented form. Fragmentedinsert regions can be obtained by mechanical force or enzymaticfragmentation methods. The fragmented insert regions can be generatedusing procedures that yield a population of fragments having overlappingsequences or non-overlapping sequences.

Mechanical fragmentation typically generates randomly fragmented nucleicacid molecules. Mechanical fragmentation methods include mechanicalshearing such as fluid shear, constant shear and pulsatile shear.Mechanical fragmentation methods also include mechanical stressincluding sonication, nebulization and acoustic cavitation.

Enzymatic fragmentation procedures can be conducted under conditionssuitable to generate randomly or non-randomly fragmented nucleic acidmolecules. For example, restriction enzyme digestion can be conducted tocompletion to generate non-randomly fragmented nucleic acid molecule.Alternatively, partial or incomplete restriction enzyme digestion can beconducted to generate randomly-fragmented nucleic acid molecules.Enzymatic fragmentation using restriction enzymes includes any one orany combination of two or more restriction enzymes selected from a groupconsisting of type I, type II, type IIs, type IIB, type III, or type IVrestriction enzymes. Enzymatic fragmentation include use of anycombination of a nicking restriction endonuclease, endonuclease and/orexonuclease.

Fragments of the insert region can be generated with PCR usingsequence-specific primers that hybridize to target regions in genomicDNA samples to generate insert regions having known fragment lengths andsequences.

Targeted genome fragmentation methods using CRISPR/Cas9 can be used togenerate fragmented insert regions.

Fragments of the insert portion can also be generated using atransposase-based tagmentation method using NEXTERA (from Epicentre).

The insert region can be single stranded or double stranded. The ends ofthe double stranded insert region can be blunt-ended, or have a 5′overhang or a 3′ overhang end, or any combination thereof. One or bothends of the insert region can be subjected to an enzymatic tailingreaction to generate a non-template poly-A tail by employing a terminaltransferase reaction. The ends of the insert region can be compatiblefor joining to at least one adaptor.

The insert region can be joined at one or both ends to at least oneadaptor. Covalent linkage between an insert region and adaptor(s) can beachieved with a DNA or RNA ligase. Exemplary DNA ligases that can ligatedouble stranded DNA molecules include T4 DNA ligase and T7 DNA ligase.An adaptor sequence can be appended to an insert sequence by PCR using atailed primer having 5′ region carrying an adaptor sequence and a 3′region that is complementary to a portion of the insert sequence. Anadaptor sequence can be appended to an insert sequence which is flankedone both sides with first and second adaptor sequences by PCR using atailed primer having 5′ region carrying a third adaptor sequence and a3′ region that is complementary to a portion of the first or secondadaptor sequence.

Adaptors comprise DNA or RNA or analogs thereof, or chimeric DNA/RNA.Adaptors can include at least one ribonucleoside residue. Adaptors canbe single-stranded, double-stranded, or have single-stranded and/ordouble-stranded portions.

Adaptors can be configured to be linear, stem-looped, hairpin, orY-shaped forms. Adaptors can be any length, including 4-100 nucleotidesor longer.

Adaptors can have blunt ends, overhang ends, or a combination of both.Overhang ends include 5′ overhang and 3′ overhang ends. The 5′ end of asingle-stranded adaptor, or one strand of a double-stranded adaptor, canhave a 5′ phosphate group or lack a 5′ phosphate group. Adaptors caninclude a 5′ tail that does not hybridize to a target polynucleotide(e.g., tailed adaptor), or adaptors can be non-tailed.

An adaptor can include a sequence (e.g., a universal adaptor sequence)that is complementary to at least a portion of a primer, such as anamplification primer, a sequencing primer, an immobilized surfacecapture primer or a soluble primer.

Adaptors can include a random sequence or degenerate sequence. Adaptorscan include at least one inosine residue.

Adaptors can be synthesized to include nucleotide analogs or modifiednucleotide linkages that confer certain properties, including resistanceto enzymatic degradation, or increased thermal stability. Examples ofnucleotide analogs and modified nucleotide linkages that inhibitnuclease digestion include phosphorothioate, 2′-O-methyl RNA, inverteddT, or 2′3′ dideoxy-dT. Adaptors that include locked nucleic acids (LNA)have increased thermal stability. Adaptors can include at least onephosphorothioate, phosphorothiolate and/or phosphoramidate linkage.

Adaptors can include at least one restriction enzyme recognitionsequence, including any one or any combination of two or more selectedfrom a group consisting of type I, type II, type III, type IV, type Hsor type IIB.

Adaptors can include a sample barcode sequence which is used todistinguish polynucleotides (e.g., insert sequences) from differentsample sources in a multiplex assay. Adaptors can include a uniqueidentification sequence (e.g., a molecular tag, unique molecular index,UMI) that is used to uniquely identify an individual nucleic acidmolecule (e.g., insert sequence) to which the adaptor is appended in apopulation of other nucleic acid molecules joined to otherdistinguishing unique identification sequence adaptors.

In some embodiments, a single stranded nucleic acid library molecule canserve as a template molecule for sequencing analysis. In someembodiments, a complementary strand of the nucleic acid library moleculecan be synthesized and the complementary strand can serve as a templatemolecule for sequencing analysis. In some embodiments, a single strandednucleic acid library molecule can be amplified and the resultingamplicon strands can serve as template molecules for sequencinganalysis. In some embodiments, double-stranded library molecules can bedenatured or subjected to enzymatic treatment to generate a singlestranded library molecule which can be subjected to binding and/orsequencing analysis.

In some embodiments, a single stranded circular library molecule can behybridized to an amplification primer (e.g., a surface primer) andsubjected to a primer extension reaction to generate a complementaryconcatemer molecule, where the concatemer molecule serves as a templatemolecule for sequencing analysis. The sequencing analysis of theconcatemer molecule can generate a complementary strand which can inturn serve as a template molecule for sequencing analysis. In someembodiments, a copy of the complementary strand can serve as a templatemolecule for sequencing analysis.

Circularization of Library Molecules

Individual linear library molecules comprise an insert sequence flankedat both ends with at least one adaptor sequence. A population of singlestranded linear library molecules can be circularized to generatecovalently closed circular library molecules. For example, the ends ofthe single-stranded linear library molecules can undergo intramolecularligation using a single-stranded ligase (e.g., CircLigase from Epicentreor Lucigen).

In some embodiments, circular DNA molecules can be generated using aprotelomerase instead of a nucleic acid ligase. Protelomerase enzymesidentifies a enzyme recognition sequence within a nucleic acid molecule,cleaves the enzyme recognition sequence to generate an end having a 5′and 3′ exposed cleavage ends, rejoins 5′ and 3′ cleavage ends of asingle exposed end at the enzyme recognition site to form a singlelinear molecule from the cleaved 5′ and 3′ ends. When this reaction isperformed on both ends of a double-stranded nucleic acid molecule havingthe enzyme recognition sequence at each end, the result is a circularnucleic acid molecule. An adaptor carrying the enzyme recognitionsequence can be joined to both ends of the double-stranded DNA moleculevia ligation or PCR using tailed PCR primers. A number of enzymes orenzyme combinations are compatible with this reaction, including aprotelomerase. One type of protelomerase is TelN protelomerase, such asthat from E. coli phage NI.

In some embodiments, a population of double-stranded linear librarymolecules can be circularized to generate circular library molecules.Individual linear library molecules comprise an insert sequence flankedat both ends with at least one adaptor sequence. In some embodiments,the population of linear library molecules can be contacted with anenzyme that catalyzes 5′ phosphorylation of the ends of the linearmolecules, such as for example T4 polynucleotide kinase. In someembodiments, the population of linear molecules having blunt ends can becontacted with a ligase enzyme for intramolecular ligation, where theligase enzyme comprises T3 or T4 DNA ligase. In some embodiments, thepopulation of linear molecules have overhang ends (e.g., sticky ends)can be contacted with a T7 DNA ligase to generate circular molecules. Insome embodiments, the linear library molecules can be reacted with theT4 polynucleotide kinase enzyme and the ligase enzyme eithersequentially or simultaneously to generate circular molecules. Thenon-circular molecules can be degraded using at least one exonucleaseenzyme, such as for example T7 exonuclease and/or exonuclease I (e.g.,thermolabile exonuclease I).

In some embodiments, a padlock probe workflow can be used to generatesingle stranded circular molecules. Typically, in the padlock probe thearrangement of the insert sequence (sequence of interest) and adaptorsdiffers from a standard linear library molecule. In some embodiments,that padlock probe comprises a single-stranded linear oligonucleotidehaving a 5′ portion, an internal linker portion, and a 3′ portion. The5′and 3′ portions each comprise a portion of a target sequence ofinterest. The 5′ and 3′ portions are separately complementary to atarget sequence of interest (e.g., a contiguous target sequence ofinterest), while the linker portion is designed to have little or nocomplementarity to the target sequence. The 5′ and 3′ ends of thepadlock probe can hybridize to adjacent positions on the target nucleicacid molecule to form an open circularized molecule with a nick betweenthe hybridized 5′ and 3′ ends. The nick can be ligated to generate acovalently close circular molecule. Alternatively, the 5′ and 3′ ends ofthe padlock probe can hybridize to adjacent positions on the targetnucleic acid molecule to form an open circularized molecule with a gapbetween the hybridized 5′ and 3′ ends. The gap can be subject to apolymerase-mediated filled-in reaction to form a nick, and the nick canbe ligated to generate a covalently close circular molecule. Thecovalently closed circular molecule can be subjected to a rolling circleamplification reaction to generate a concatemer having tandem repeatregions containing the sequence of interest. The internal linker portioncan be engineered to include one or more universal adaptor sequences,barcode adaptors or unique identifier adaptors.

In some embodiments, a population of the single stranded linear librarymolecules can be circularized to generate single stranded circularlibrary molecules. Individual single stranded linear library moleculescomprise an insert sequence flanked at both ends with at least oneadaptor sequence. For example, the single stranded linear librarymolecules comprises the arrangement: 5′—first surface primer bindingsequence (SP1); first sequencing primer binding sequence (Seq1); insertsequence (sequence of interest); second sequencing primer bindingsequence (Seq2); second surface primer binding sequence (SP2)-3′ (e.g.,see FIG. 53 left top schematic). The population of double strandedlinear library molecules can be denatured to generate single strandedlinear library molecules. The single stranded linear library moleculescan be hybridized to partially double stranded splint molecules togenerate circular library molecules with gaps. The partially doublestranded splint molecules comprise double stranded oligonucleotideshaving a first splint strand (long strand) and a second splint strand(short strand) (e.g., see FIG. 53 , left bottom schematic). In someembodiments, the first splint strand comprises: (i) a left sequence thathybridizes to a first surface primer binding sequence (SP1′); (ii) aninternal portion that hybridizes to the second splint strand; and (iii)a right sequence that hybridizes to a second surface primer bindingsequence (SP2′) (e.g., see FIG. 53 ). In some embodiments, the internalportion of the first splint strand comprises a complementary sequence ofa sample barcode and/or a unique identification sequence (N′N′N′). Insome embodiments, the second splint strand comprises a sequence thathybridizes to the internal portion of the first splint strand. Thesecond splint strand can include a sample barcode and/or uniqueidentification sequence (NNN). The insert sequence of interest does nothybridize to the first or second splint strands (FIG. 53 ).

A single stranded library molecule hybridizes to a double strandedsplint molecule to generate a library-splint complex having two nicks(FIG. 54 ). The first nick is located between the 5′ end of the librarymolecule and the 3′ end of the second splint strand. The second nick islocated between the 3′ end of the library molecule and the 5′ end of thesecond splint strand (FIG. 54 , left schematic).

The library-splint complexes can be reacted with T4 polynucleotidekinase and a ligase (e.g., T7 DNA ligase) either sequentially orsimultaneously, to (i) phosphorylate the 5′ end of the library molecule,the 5′ end of the first splint strand, and the 5′ end of the secondsplint strand, and to (ii) close the first and second nicks by enzymaticligation, thereby generating a single stranded covalently closedcircular library molecule which is hybridized to the second splintstrand (FIG. 54 , center schematic). The non-circular molecules and thesecond splint strands can be degraded using at least one exonucleaseenzyme, such as for example T7 exonuclease and/or exonuclease I (e.g.,thermolabile exonuclease I). The remaining single stranded circularlibrary molecules can be subjected to a rolling circle amplificationreaction, either in-solution or on-support, using the 3′ end of thelinear first splint strand (FIG. 54 , right schematic). Alternatively,the first splint strand can be removed and the closed circular moleculecan be hybridized with a soluble amplification primer which can be usedto initiate a rolling circle amplification reaction. In another example,the first splint strand can be removed and the closed circular moleculecan be hybridized with an immobilized surface primer on a support andthen subjected to a rolling circle amplification reaction. The surfaceprimers that are immobilized to the support lack a nucleotide having ascissile moiety. For example, the surface primers lack uridine,8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine.

Compaction Oligonucleotides

Any of the on-support or in-solution rolling circle amplificationreactions described herein can include a plurality of compactionoligonucleotides which can compact the size and/or shape of theconcatemer into a nanoball. The compaction oligonucleotide is asingle-stranded nucleic acid molecule comprising DNA or analogs thereof.The compaction oligonucleotide can cross-hybridize to portions of theconcatemer molecule. The compaction oligonucleotide comprises twoidentical sequences separated by a short linker sequence, where the twoidentical sequences are reverse-complementary to a portion of theconcatemer. The compaction oligonucleotide can be any length, forexample 20-100 nucleotides. The two identical sequence regions of thecompaction oligonucleotide can hybridize to the concatemer to pulltogether distal portions of the concatemer causing compaction of theconcatemer to form a nanoball. In some embodiments, the compactionoligonucleotide is resistant to 3′ exonuclease degradation and/orsingle-stranded endonuclease degradation. In some embodiments, thecompaction oligonucleotide comprises any one or any combination of twoor more of: 3′ terminal end phosphorylation; at least two 3′ terminalend nucleotides having a phosphorothioate bond therebetween; at leastone 3′ terminal end nucleotide having a 2′-O-methyl moiety; and/or atleast one 3′ terminal nucleotide having a 2′ fluoro base.

The compaction oligonucleotides can include at least one region havingconsecutive guanines. For example, the compaction oligonucleotides caninclude at least one region having 2, 3, 4, 5, 6 or more consecutiveguanines. In some embodiments, the compaction oligonucleotides comprisefour consecutive guanines which can form a guanine tetrad structure (seeFIG. 116A). The guanine tetrad structure can be stabilized via Hoogsteenhydrogen bonding. The guanine tetrad structure can be stabilized by acentral cation including potassium, sodium, lithium, rubidium or cesium.

In some embodiments, the rolling circle amplification reaction can beconducted with circular library molecules comprising a sequence ofinterest and a universal binding sequence for a compactionoligonucleotide, and further comprising any one or any combination oftwo or more of: (i) two or more copies of a universal binding sequencefor a soluble forward sequencing primer, (ii) two or more copies of auniversal binding sequence for a soluble reverse sequencing primer,(iii) two or more copies of a universal binding sequence for animmobilized first surface primer, (iv) two or more copies of a universalbinding sequence for an immobilized second surface primer, (v) two ormore copies of a universal binding sequence for a first solubleamplification primer, (vi) two or more copies of a universal bindingsequence for a second soluble amplification primer, (vii) two or morecopies of a sample barcode sequence and/or (viii) two or more copies ofa unique molecular index sequence.

The rolling circle amplification reaction can be conducted in thepresence of a plurality of compaction oligonucleotides having at leastfour consecutive guanines. The resulting concatemers comprise repeatcopies of the universal binding sequence for the compactionoligonucleotide. At least one compaction oligonucleotide can form aguanine tetrad (FIG. 116A) and hybridize to the universal bindingsequences for the compaction oligonucleotide, and the resultingconcatemer can fold to form an intramolecular G-quadruplex structure(FIG. 116B). The concatemers can self-collapse to form compactnanoballs. Formation of the guanine tetrads and G-quadruplexes in thenanoballs may increase the stability of the nanoballs to retain theircompact size and shape which can withstand repeated flows of reagentsfor conducting any of the pairwise sequencing workflows describedherein.

On-Support Rolling Circle Amplification

The present disclosure provides pairwise sequencing compositions andmethods which employ on-support rolling circle amplification to generatea plurality of immobilized single stranded nucleic acid concatemertemplate molecules. In some embodiments, the on-support rolling circleamplification reaction comprises: (1) hybridizing a plurality ofsingle-stranded covalently closed circular nucleic acid librarymolecules to the plurality of immobilized surface primers therebygenerating a plurality of immobilized covalently closed circular nucleicacid library molecules; (2) forming a plurality of nucleotide-polymerasecomplexes by contacting the plurality of immobilized covalently closedcircular nucleic acid library molecules with (i) a plurality ofpolymerases having strand displacement activity, (ii) a plurality ofnucleotides comprising a mixture of dATP, dGTP, dCTP, dTTP and anucleotide analog having a scissile moiety, and (iii) a catalyticdivalent cation that mediates nucleotide binding to thenucleotide-polymerase complexes and promotes nucleotide incorporation(e.g., magnesium and/or manganese), wherein the contacting is conductedunder a condition suitable for binding individual polymerases to animmobilized covalently closed circular nucleic acid library molecule toform complexed polymerases and the condition is suitable for bindingindividual complexed polymerases to a nucleotide or nucleotide analog toform the nucleotide-polymerase complexes. In some embodiments, in thenucleotide mixture of the plurality of nucleotides, the nucleotidehaving the scissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine(e.g., 8oxoG) or deoxyinosine; and (3) conducting a nucleotidepolymerization reaction (e.g., rolling circle amplification reaction)under an isothermal temperature condition to generate a plurality ofimmobilized single stranded concatemer template molecules.

In some embodiments, the rolling circle amplification reaction lacks aplurality of compaction oligonucleotides, or the rolling circleamplification reaction further comprises a plurality of compactionoligonucleotides that can hybridize to portions of the concatemer tocollapse the concatemer into a more compact shape and size. Thecompaction oligonucleotide is a single-stranded nucleic acid moleculehaving two identical sequences separated by a short linker sequence,where the two identical sequences are reverse-complementary to a portionof the concatemer. The compaction oligonucleotide can be any length, forexample 20-100 nucleotides. The two identical sequence regions of thecompaction oligonucleotide can hybridize to the concatemer to pulltogether distal portions of the concatemer causing compaction of theconcatemer. In some embodiments, the compaction oligonucleotide isresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the compactionoligonucleotide comprises any one or any combination of two or more of:3′ terminal end phosphorylation; at least two 3′ terminal endnucleotides having a phosphorothioate bond therebetween; at least one 3′terminal end nucleotide having a 2′-O-methyl moiety; and/or at least one3′ terminal nucleotide having a 2′ fluoro base. In some embodiments, thecompaction oligonucleotides can be included in step (2) or (3).

In some embodiments, the plurality of polymerases having stranddisplacement activity comprise phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-)DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase canbe wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), orvariant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific),or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, the rolling circle amplification reaction furthercomprises at least one accessory protein or enzyme, including helicase,single-stranded binding (SSB) protein, or recombinase (e.g., T4 uvsX)and/or recombinase accessory factor (e.g., T4 uvsY or T4 gp32).

In some embodiments, the rolling circle amplification reaction can beconducted at an isothermal temperature of about 30, 31, 32, 33, 34, 35,36, 37, 38, 39 or 40° C.

In some embodiments, the concatemer can contain at least 2, 5, 10, 50,100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10,000, or more tandem copies of repeat units which include the of thesequence of interest (or complementary sequence thereof) and any adaptorsequences (or complementary sequence thereof) present in the originalcovalently closed circular nucleic acid library molecules.

In some embodiments, the rolling circle amplification (RCA) reaction canbe followed by a multiple displacement amplification (MDA) reaction or aflexing amplification reaction. In some embodiments, the RCA reaction isnot followed by a multiple displacement amplification reaction or aflexing amplification reaction. Exemplary MDA and flexing reactions aredescribed below.

On-Support Two-Stage Rolling Circle Amplification

Trapped Nucleotide-Polymerase Complexes

The present disclosure provides pairwise sequencing compositions andmethods which employ an on-support two-stage rolling circleamplification reactions to generate a plurality of immobilized singlestranded nucleic acid concatemer template molecules. The two-stagerolling circle amplification methods described herein employsnon-catalytic and then catalytic divalent cations to synchronize therolling circle amplification events on a surface/support and generateconcatemers. The two-stage rolling circle amplification reaction can befollowed by a relaxant condition and a flexing amplification reactionwhich generates new concatemers from the existing concatemers. Together,these amplification methods generate highly compact nanoballs containinghigh copy number of the target sequence which improves sequencing signalintensity.

In some embodiments, the on-support two-stage rolling circleamplification reaction comprises: (1) hybridizing a plurality ofsingle-stranded covalently closed circular nucleic acid librarymolecules to the plurality of immobilized surface primers therebygenerating a plurality of immobilized covalently closed circular nucleicacid library molecules; (2) forming a plurality of trappednucleotide-polymerase complexes by contacting the plurality ofimmobilized covalently closed circular nucleic acid library moleculeswith (i) a plurality of polymerases having strand displacement activity,(ii) a first plurality of nucleotides comprising a mixture of dATP,dGTP, dCTP, dTTP and a nucleotide analog having a scissile moiety, and(iii) a non-catalytic divalent cation that mediates nucleotide bindingto the trapped nucleotide-polymerase complexes but not nucleotideincorporation (e.g., strontium or barium), wherein the contacting isconducted under a condition suitable for binding individual polymerasesto an immobilized covalently closed circular nucleic acid librarymolecule to form complexed polymerases and the condition is suitable forbinding individual complexed polymerases to a nucleotide or nucleotideanalog to form the trapped nucleotide-polymerase complexes. In someembodiments, in the nucleotide mixture of the first plurality ofnucleotides, the nucleotide having the scissile moiety comprisesuridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. Therolling circle amplification reaction further comprises: (3) conductinga nucleotide polymerization reaction by contacting the plurality oftrapped nucleotide-polymerase complexes with (i) at least one divalentcation that mediates nucleotide binding and mediates nucleotideincorporation (e.g., magnesium and/or manganese), and (ii) a secondplurality of nucleotides comprising a mixture of dATP, dGTP, dCTP, dTTPand a nucleotide analog having a scissile moiety, under a conditionsuitable for conducting an isothermal rolling circle amplificationreaction to generate a plurality of immobilized single strandedconcatemer template molecules. In some embodiments, in the nucleotidemixture of the second plurality of nucleotides, the nucleotide analoghaving the scissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine(e.g., 8oxoG) or deoxyinosine.

In some embodiments, the two-stage rolling circle amplification reactionlacks a plurality of compaction oligonucleotides, or the rolling circleamplification reaction further comprises a plurality of compactionoligonucleotides that can hybridize to portions of the concatemer tocollapse the concatemer into a more compact shape and size. Thecompaction oligonucleotide is a single-stranded nucleic acid moleculehaving two identical sequences separated by a short linker sequence,where the two identical sequences are reverse-complementary to a portionof the concatemer. The compaction oligonucleotide can be any length, forexample 20-100 nucleotides. The two identical sequence regions of thecompaction oligonucleotide can hybridize to the concatemer to pulltogether distal portions of the concatemer causing compaction of theconcatemer. In some embodiments, the compaction oligonucleotide isresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the compactionoligonucleotide comprises any one or any combination of two or more of:3′ terminal end phosphorylation; at least two 3′ terminal endnucleotides having a phosphorothioate bond therebetween; at least one 3′terminal end nucleotide having a 2′-O-methyl moiety; and/or at least one3′ terminal nucleotide having a 2′ fluoro base. In some embodiments, thecompaction oligonucleotides can be included in step (2) or (3).

In some embodiments, in the trapped nucleotide-polymerase mixture ofstep (2), the plurality of polymerases having strand displacementactivity comprise phi29 DNA polymerase, large fragment of Bst DNApolymerase, large fragment of Bsu DNA polymerase, and Bca (exo-) DNApolymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase,M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or DeepVent DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNApolymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

In some embodiments, the on-support two-stage rolling circleamplification reaction further comprises at least one accessory proteinor enzyme, including helicase, single-stranded binding (SSB) protein, orrecombinase (e.g., T4 uvsX) and/or recombinase accessory factor (e.g.,T4 uvsY or T4 gp32).

In some embodiments, the on-support two-stage rolling circleamplification reaction can be conducted at an isothermal temperature ofabout 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40° C.

In some embodiments, the concatemer can contain at least 2, 5, 10, 50,100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10,000, or more tandem copies of repeat units which include the of thesequence of interest (or complementary sequence thereof) and any adaptorsequences (or complementary sequences thereof) present in the originalcovalently closed circular nucleic acid library molecules.

In some embodiments, the on-support two-stage rolling circleamplification (RCA) reaction with trapped nucleotide-polymerasecomplexes can be followed by a multiple displacement amplification (MDA)reaction or a flexing amplification reaction. In some embodiments, theRCA reaction with trapped nucleotide-polymerase complexes is notfollowed by a multiple displacement amplification reaction or a flexingamplification reaction. Exemplary MDA and flexing reactions aredescribed below.

In-Solution Initiated Two-Stage Rolling Circle Amplification

Trapped Nucleotide-Polymerase Complexes

The present disclosure provides pairwise sequencing compositions andmethods which employ an in-solution two-stage rolling circleamplification reactions to generate a plurality of single strandednucleic acid concatemer template molecules. The two-stage rolling circleamplification methods described herein employs non-catalytic and thencatalytic divalent cations to synchronize the rolling circleamplification events in-solution and generate concatemers. Theconcatemers generated in-solution using the two-stage rolling circleamplification reaction can be distributed onto a support having surfaceprimers immobilized thereon. The concatemers can hybridize to theimmobilized surface primers, and the rolling circle amplificationreaction can continue on the support. These amplification methodsgenerate highly compact nanoballs containing high copy number of thetarget sequence which improves sequencing signal intensity.

In some embodiments, the in-solution two-stage rolling circleamplification reaction comprises: (1) forming templates-primer duplexesby hybridizing in-solution circular nucleic acid library molecules withsoluble first amplification primers; (2) forming a plurality of trappednucleotide-polymerase complexes by contacting the template-primerduplexes with (i) a plurality of polymerases having strand displacingactivity, (ii) a first plurality of nucleotides comprising a mixture ofdATP, dGTP, dCTP, dTTP and a nucleotide analog having a scissile moiety,and (iii) a non-catalytic divalent cation that mediates nucleotidebinding to the trapped nucleotide-polymerase complexes but notnucleotide incorporation (e.g., strontium or barium), wherein thecontacting is conducted under a condition suitable for bindingindividual polymerases to an immobilized covalently closed circularnucleic acid library molecule to form complexed polymerases and thecondition is suitable for binding individual complexed polymerases to anucleotide or nucleotide analog to form the trappednucleotide-polymerase complexes. In some embodiments, in the nucleotidemixture of the first plurality of nucleotides, the nucleotide having thescissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine (e.g.,8oxoG) or deoxyinosine. The rolling circle amplification reactionfurther comprises: (3) conducting a nucleotide polymerization reactionby contacting the plurality of trapped nucleotide-polymerase complexeswith (i) at least one divalent cation that mediates nucleotide bindingand mediates nucleotide incorporation (e.g., magnesium and/ormanganese), and (ii) a second plurality of nucleotides comprising amixture of dATP, dGTP, dCTP, dTTP and a nucleotide analog having ascissile moiety, under a condition suitable for conducting an isothermalrolling circle amplification reaction to generate a plurality ofimmobilized single stranded concatemer template molecules. In someembodiments, in the nucleotide mixture of the second plurality ofnucleotides, the nucleotide analog having the scissile moiety comprisesuridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. Thein-solution RCA reaction can be conducted for an amount of time suitableto generate a plurality of concatemers hybridized to their respectivecircular library molecules which are then distributed onto the supporthaving a plurality of surface primers immobilized thereon. Thein-solution RCA reaction can be conducted for a very short period oftime, for a moderate period of time, or for longer periods of time,prior to distributing onto the support.

In some embodiments, the two-stage rolling circle amplification reactionlacks a plurality of compaction oligonucleotides, or the rolling circleamplification reaction further comprises a plurality of compactionoligonucleotides that can hybridize to portions of the concatemer tocollapse the concatemer into a more compact shape and size. Thecompaction oligonucleotide is a single-stranded nucleic acid moleculehaving two identical sequences separated by a short linker sequence,where the two identical sequences are reverse-complementary to a portionof the concatemer. The compaction oligonucleotide can be any length, forexample 20-100 nucleotides. The two identical sequence regions of thecompaction oligonucleotide can hybridize to the concatemer to pulltogether distal portions of the concatemer causing compaction of theconcatemer. In some embodiments, the compaction oligonucleotide isresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the compactionoligonucleotide comprises any one or any combination of two or more of:3′ terminal end phosphorylation; at least two 3′ terminal endnucleotides having a phosphorothioate bond therebetween; at least one 3′terminal end nucleotide having a 2′-O-methyl moiety; and/or at least one3′ terminal nucleotide having a 2′ fluoro base. In some embodiments, thecompaction oligonucleotides can be included in step (2) or (3).

In some embodiments, in the trapped nucleotide-polymerase mixture ofstep (2), the plurality of polymerases having strand displacementactivity comprise phi29 DNA polymerase, large fragment of Bst DNApolymerase, large fragment of Bsu DNA polymerase, and Bca (exo-) DNApolymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase,M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or DeepVent DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNApolymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNApolymerase (e.g., from Thermo Fisher Scientific), or chimeric QualiPhiDNA polymerase (e.g., from 4basebio).

In some embodiments, the in-solution two-stage rolling circleamplification reaction further comprises at least one accessory proteinor enzyme, including helicase, single-stranded binding (SSB) protein, orrecombinase (e.g., T4 uvsX) and/or recombinase accessory factor (e.g.,T4 uvsY or T4 gp32).

In some embodiments, the in-solution two-stage rolling circleamplification reaction can be conducted at an isothermal temperature ofabout 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40° C.

In some embodiments, the concatemer can contain at least 2, 5, 10, 50,100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10,000, or more tandem copies of repeat units which include the of thesequence of interest (or complementary sequence thereof) and any adaptorsequences (or complementary sequences thereof) present in the originalcovalently closed circular nucleic acid library molecules.

In some embodiments, the in-solution two-stage rolling circleamplification (RCA) reaction with trapped nucleotide-polymerasecomplexes can be followed by an in-solution multiple displacementamplification (MDA) reaction or an on-support flexing amplificationreaction. In some embodiments, the in-solution RCA reaction with trappednucleotide-polymerase complexes is not followed by a multipledisplacement amplification reaction or a flexing amplification reaction.Exemplary MDA and flexing reactions are described below.

Multiple Displacement Amplification Reaction with Random-SequencePrimers

In some embodiments, the on-support or the in-solution two-stage rollingcircle amplification methods can optionally be followed by a multipledisplacement amplification reaction which employs random-sequencesoluble primers. The multiple displacement amplification reactioncomprises: (1) forming a multiple displacement amplification (MDA)reaction mixture by contacting the plurality of immobilized singlestranded concatemer template molecules with (i) a second plurality ofpolymerases having strand displacement activity, and (ii) a plurality ofsoluble amplification primers wherein individual amplification primersin the plurality are exonuclease-resistant and have a 3′ OH extendibleend and comprise a random sequence that can hybridize to a portion ofthe concatemer template molecules, (iii) a third plurality ofnucleotides comprising a mixture of dATP, dGTP, dCTP, dTTP andoptionally a nucleotide analog having a scissile moiety, and (iv) atleast one divalent cation that mediates nucleotide binding and mediatesnucleotide incorporation (e.g., magnesium and/or manganese); and (2)conducting an isothermal multiple displacement amplification (MDA)reaction to generate a plurality of immobilized branched concatemers. Insome embodiments, the nucleotide analog having the scissile moietycomprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) ordeoxyinosine.

In some embodiments, in the multiple displacement amplification (MDA)reaction mixture, the second plurality of polymerases having stranddisplacement activity comprises phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-)DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase canbe wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), orvariant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific),or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, in the multiple displacement amplification (MDA)reaction mixture, the plurality of amplification primers comprisesingle-stranded nucleic acid primers having a length of about 5-25nucleotides. In some embodiments, the plurality of soluble amplificationprimers comprise non-protected single-stranded nucleic acid primers. Insome embodiments, the plurality of soluble amplification primerscomprise protected single-stranded nucleic acid primers that areresistant to 3′ exonuclease degradation and/or single-strandedendonuclease degradation. In some embodiments, the plurality of solubleamplification primers comprise any one or any combination of two or moreof: 3′ terminal end phosphorylation; at least two 3′ terminal endnucleotides having a phosphorothioate bond therebetween; at least one 3′terminal end nucleotide having a 2′-O-methyl moiety; and/or at least one3′ terminal nucleotide having a 2′ fluoro base. In some embodiments, theplurality of soluble amplification primers comprise a population ofprimers having the same length, for example a length of 6 or 9nucleotides. In some embodiments, the plurality of soluble amplificationprimers comprise a population of primers having a mixture of differentlengths, for example a mixture comprising 6-mer and 9-mer primers. Insome embodiments, the plurality of soluble amplification primerscomprise a mixture of primers having random sequences including up to 4⁶different sequences (e.g., for the 6-mers) or 4⁹ different sequences(e.g., for the 9-mers).

In some embodiments, the multiple displacement amplification (MDA)reaction mixture can further comprise at least one accessory protein orenzyme, including helicase, single-stranded binding (SSB) protein, orrecombinase (e.g., T4 uvsX) and/or recombinase accessory factor (e.g.,T4 uvsY or T4 gp32).

In some embodiments, the multiple displacement amplification (MDA)reaction can be conducted at an isothermal temperature of about 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45° C.

Multiple Displacement Amplification Reaction with Primase-Polymerase

In some embodiments, the on-support or the in-solution two-stage rollingcircle amplification methods can optionally be followed by a multipledisplacement amplification reaction which employs a primase-polymeraseenzyme. The multiple displacement amplification reaction comprises: (1)forming a multiple displacement amplification (MDA) reaction mixture bycontacting the plurality of immobilized single stranded concatemertemplate molecules with (i) a second plurality of polymerases havingstrand displacement activity, (ii) a plurality of DNA primase-polymeraseenzymes, (iii) a third plurality of nucleotides comprising a mixture ofdATP, dGTP, dCTP, dTTP and optionally a nucleotide analog having ascissile moiety, and (iv) at least one divalent cation that mediatesnucleotide binding and mediates nucleotide incorporation (e.g.,magnesium and/or manganese), and (2) conducting an isothermal multipledisplacement amplification (MDA) reaction to generate a plurality ofimmobilized branched concatemers. In some embodiments, the multipledisplacement amplification reaction is conducted without addedamplification primers (e.g., a primerless reaction). In someembodiments, the nucleotide analog having the scissile moiety comprisesuridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine.

In some embodiments, in the multiple displacement amplification (MDA)reaction mixture, the second plurality of polymerases having stranddisplacement activity comprises phi29 DNA polymerase, large fragment ofBst DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-)DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5polymerase, M-MuLV reverse transcriptase, HIV viral reversetranscriptase, or Deep Vent DNA polymerase. The phi29 DNA polymerase canbe wild type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), orvariant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific),or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, the plurality of DNA primase-polymerase enzymescomprise an enzyme from Thermus thermophilus HB27 (e.g., Tth PrimPolenzyme).

In some embodiments, the multiple displacement amplification (MDA)reaction mixture further comprises at least one accessory protein orenzyme, including helicase, single-stranded binding (SSB) protein, orrecombinase (e.g., T4 uvsX) and/or recombinase accessory factor (e.g.,T4 uvsY or T4 gp32).

In some embodiments, the multiple displacement amplification (MDA)reaction can be conducted at an isothermal temperature of about 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45° C.

Flexing Amplification

In some embodiments, the immobilized single stranded concatemer templatemolecules that are generated by the on-support or the in-solutiontwo-stage rolling circle amplification methods can optionally besubjected to a two stage flexing amplification method on the support,which comprises contacting the immobilized single stranded concatemertemplate molecules with nucleic acid relaxing agents (first stage) andthen conducting a flexing amplification reaction during the secondstage. Without wishing to be bound by theory, it is postulated that thenucleic acid relaxing agent(s) can disrupt hydrogen bonding (e.g.,denaturation) in the plurality of immobilized concatemer templatemolecules which causes the structure of the nucleic acid concatemers torelax and increases the number of new duplex formations between theimmobilized surface primers and portions of the nucleic acidconcatemers, thereby increasing the opportunity to generate newconcatemers from the duplexed immobilized surface primers. The newconcatemers can be generated during the flexing amplification reaction.The inclusion of the relaxing agents can cause nucleic acid denaturationwithout use of denaturation temperatures or denaturation chemicals.

In general, the amplification method comprises: (1) conductingon-support rolling circle amplification (e.g., two-stage RCA) togenerate a plurality of immobilized single stranded concatemer templatemolecules; (2) forming a relaxant reaction mixture with a temperatureramp-up and ramp-down followed by washing; (3) forming a flexingamplification reaction mixture; (4) conducting a flexing amplificationreaction on the support (e.g., with no added soluble primers) togenerate a plurality of double-stranded concatemers; (5) washing; and(6) repeating steps (2)-(5) at least once.

In some embodiments, the flexing amplification method comprises: (1)conducting the on-support rolling circle amplification therebygenerating a plurality of immobilized single stranded concatemertemplate molecules; (2) forming a relaxant reaction mixture under acondition suitable for increasing the number of duplex formationsbetween the immobilized surface primers (e.g., that are not yethybridized to a covalently closed circular nucleic acid library moleculeor not covalently linked to a single stranded concatemer templatemolecule) and portions of the single-stranded concatemer templatemolecules, by contacting the single-stranded concatemer templatemolecules with at least one nucleic acid relaxing reagent under atemperature ramp-up condition, a relaxant incubation condition, and atemperature ramp-down condition, and followed by a washing step; (3)forming a flexing amplification reaction mixture by contacting thesingle-stranded concatemers template molecules with (i) a plurality ofpolymerases having strand displacement activity, (ii) a plurality ofnucleotides wherein the concentration of the plurality of nucleotides isat least at the threshold concentration, or exceeds the thresholdconcentration, to promote nucleotide polymerization, (iii) a divalentcation that mediates nucleotide binding and mediates nucleotidepolymerization (e.g., magnesium and/or manganese), wherein the flexingreaction mixture contains no added soluble amplification primers; (4)conducting a flexing amplification reaction under a condition suitablefor generating a plurality of immobilized double-stranded concatemers byconducting a temperature ramp-up condition, an amplification incubationcondition, and a temperature ramp-down condition, wherein individualconcatemers in the plurality of double-stranded nucleic acid concatemershave two or more tandem copies of repeat units which include the of thesequence of interest (or complementary sequence thereof) and any adaptorsequences (or complementary sequence thereof) present in the immobilizedsingle stranded concatemer template molecules; (5) washing theimmobilized double-stranded concatemers to removes the strand-displacingpolymerase under condition suitable to retain the immobilizeddouble-stranded concatemers; and (6) repeating steps (2)-(5) at leastonce (e.g., at least one cycle).

In some embodiments, the relaxant reaction mixture of step (2) can beformed with at least one nucleic acid relaxing agent that can disrupthydrogen bonding in the immobilized nucleic acid concatemers. Exemplaryrelaxing agents include nucleic acid denaturants, chaotropic compounds,amide compounds, aprotic compounds, primary alcohols and ethylene glycolderivatives. Chaotropic compounds comprise urea, guanidine hydrochlorideor guanidine thiocyanate. Amide compounds comprise formamide, acetamideor NN-dimethylformamide (DMF). Aprotic compounds comprise acetonitrile,DMSO (dimethyl sulfoxide), 1,4-dioxane or tetrahydrofuran. Primaryalcohols comprise 1-propanol, ethanol or methanol. Ethylene glycolderivatives comprise 1,3-propanediol, ethylene glycol, glycerol,1,2-dimethyoxyethane or 2-methoxyethanol. Other relaxing agents includesodium iodide, potassium iodide and polyamines

In some embodiments, the relaxant reaction mixture of step (2) comprisesany one or a combination of two or more of a group selected from urea,guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide,NN-dimethylformamide (DMF), acetonitrile, DMSO (dimethyl sulfoxide),1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol,1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethyoxyethane,2-methoxyethanol, sodium iodide, potassium iodide and/or polyamines.

In some embodiments, the relaxant reaction mixture of step (2) comprisesformamide and SSC. In some embodiments, the relaxant reaction mixturecomprises acetonitrile, formamide and SSC. In some embodiments, therelaxant reaction mixture comprises acetonitrile, formamide and MES(2-(4-morpholino)-ethane sulfonic acid). In some embodiments, therelaxant reaction mixture comprises acetonitrile, formamide, guanidiumhydrochloride and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid). In some embodiments, the relaxant reaction mixture comprisesacetonitrile, formamide, urea and HEPES. In some embodiments, the SSC inthe relaxant reaction mixture can be 1×, 2×, 3× or 4×.

In some embodiments, in the forming the relaxant reaction mixture ofstep (2), the temperature ramp-up condition can be conducted from about20° C. to about 70° C., the relaxant incubation condition can beconducted at a temperature of about 40-70° C., and the temperatureramp-down condition can be conducted from about 70° C. to about 20° C. Askilled artisan will recognize that the temperature ramp-up, relaxantincubation temperature, and temperature ramp-down conditions can bemodified.

In some embodiments, in the flexing amplification reaction mixture ofstep (3), the second plurality of polymerases having strand displacementactivity comprises large fragment of Bst DNA polymerase (e.g.,exonuclease minus), phi29 DNA polymerase, large fragment of Bsu DNApolymerase, and Bca (exo-) DNA polymerase, Klenow fragment of E. coliDNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viralreverse transcriptase, or Deep Vent DNA polymerase. The phi29 DNApolymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi fromExpedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo FisherScientific), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio).

In some embodiments, in the flexing amplification reaction mixture ofstep (3), the concentration (e.g., total concentration) of the thirdplurality of nucleotides can promote a nucleotide polymerizationreaction. For example, the concentration (e.g., total concentration) ofthe third plurality of nucleotides is about 0.1-10 mM.

In some embodiments, the plurality of nucleotides in the flexingamplification reaction mixture of step (3) comprise a mixture ofnucleotide comprising dATP, dGTP, dCTP, and optionally a nucleotideanalog having a scissile moiety. In some embodiments, the nucleotidehaving the scissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine(e.g., 8oxoG) or deoxyinosine.

In some embodiments, in the flexing amplification reaction mixture ofstep (3), the at least one divalent cation that mediates nucleotidebinding and mediates nucleotide polymerization comprises a catalyticdivalent cation. In some embodiments, the catalytic divalent cationcomprises magnesium and/or manganese. The concentration of the catalyticdivalent cation in the amplification reaction mixture can be about 1-20mM.

In some embodiments, the flexing amplification reaction mixture of step(3) can include at least one accessory protein or enzyme, includinghelicase, single-stranded binding (SSB) protein, or recombinase (e.g.,T4 uvsX) and/or recombinase accessory factor (e.g., T4 uvsY or T4 gp32).In some embodiments, these accessory proteins can be omitted.

In some embodiments, in the flexing amplification reaction of step (4),the temperature ramp-up condition can be conducted from about 20° C. toabout 90° C.

In some embodiments, in the flexing amplification reaction of step (4),the temperature ramp-up condition can be conducted for about 5-15seconds, or about 15-30 seconds, or about 30-45 seconds, or about 45-60seconds, or longer.

In some embodiments, in the flexing amplification reaction of step (4),the amplification incubation condition can be about 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70° C., orat a higher temperature.

In some embodiments, in the flexing amplification reaction of step (4),the amplification incubation condition can be conducted for about 30-45seconds, or about 45-60 seconds, or about 60-75 seconds, or about 75-90seconds, or longer.

In some embodiments, in the flexing amplification reaction of step (4),the temperature ramp-down condition can be conducted from about 90° C.to about 20° C.

In some embodiments, in the flexing amplification reaction of step (4),the temperature ramp-down condition can be conducted for about 5-15seconds, or about 15-30 seconds, or about 30-45 seconds, or about 45-60seconds, or longer.

In some embodiments, in the washing of step (5), the wash buffercomprises 1×SSC, or 1×SSC with cobalt hexamine.

In some embodiments, steps (2)-(5) can be repeated at least once, orrepeated up to 10 times, or repeated up to 15 times, or repeated up to20 times, or repeated up to 30 times or more.

Sequencing Methods

The present disclosure provides methods for conducting pairwisesequencing that employs a sequencing polymerase and labeled ornon-labeled chain terminating nucleotides, where the chain terminatingnucleotides include a 3′-O-azido group (or 3′-O-methylazido group) orany other type of bulky blocking group at the sugar 3′ position. Forexample, sequencing-by-synthesis (SBS) methods employ labeledchain-terminating nucleotides, and sequencing-by-binding methods (SBB)employ non-labeled chain-terminating nucleotides.

The present disclosure also provides methods for conducting pairwisesequencing using sequencing-by-avidity methods (SBA) that employs asequencing polymerase and labeled multivalent molecules and non-labeledchain terminating nucleotides.

Sequencing-by-avidity (SBA) of a template molecule (e.g., DNA templatemolecule) ideally requires (a) the detection of the n+1 base andrequires 2 or more copies of target nucleic acid sequence, two or moreprimer nucleic acid molecules that are complementary to one or moreregions of said target nucleic acid sequence and two more polymerasescontacting said composition with a multivalent molecule (e.g., apolymer-nucleotide conjugate) under conditions sufficient to allow amultivalent binding complex to be formed between said polymer-nucleotideconjugate and said two or more copies of said target nucleic acidsequence in said composition of wherein the polymer-nucleotide conjugatecomprises two or more nucleotide moieties; the detection substrates issubsequently washed away and (b) to ensure only a single incorporationoccurs, a structural modification (‘blocking group’) of the an unlabelednucleotides is required to ensure a single nucleotide incorporation butwhich then prevents any further nucleotide incorporation into thepolynucleotide chain. The blocking group must then be removable, underreaction conditions which do not interfere with the integrity of the DNAbeing sequenced. The sequencing cycle can then continue with the N+1detection of the next multivalent polymerase-conjugate-DNA complex andso on. In order to be of practical use, the avidity step requires both(a) a stable substrate to persist for long enough to image for >30 s and(b) a stepping step whereby the entire process should consist of highyielding, highly specific chemical and enzymatic steps to facilitatemultiple cycles of sequencing.

Sequencing-by-synthesis (SBS) of a template molecule (e.g., DNA templatemolecule) ideally requires the controlled (i.e. one at a time)incorporation of the correct complementary nucleotide opposite theoligonucleotide being sequenced. This allows for accurate sequencing byadding nucleotides in multiple cycles as each nucleotide residue issequenced one at a time, thus preventing an uncontrolled series ofincorporations occurring. The incorporated nucleotide is read using anappropriate label attached thereto before removal of the label moietyand the subsequent next round of sequencing. In order to ensure only asingle incorporation occurs, a structural modification (‘blockinggroup’) of the sequencing nucleotides is required to ensure a singlenucleotide incorporation but which then prevents any further nucleotideincorporation into the polynucleotide chain. The blocking group mustthen be removable, under reaction conditions which do not interfere withthe integrity of the DNA being sequenced. The sequencing cycle can thencontinue with the incorporation of the next blocked, labellednucleotide. In order to be of practical use, the entire process shouldconsist of high yielding, highly specific chemical and enzymatic stepsto facilitate multiple cycles of sequencing.

Sequencing-by-binding (SBB) a template molecule (e.g., DNA templatemolecule) requires method for sequencing a nucleic acid that includesthe steps of (a) sequentially contacting a primed template nucleic acidwith at least two separate mixtures under ternary complex stabilizingconditions, wherein the at least two separate mixtures each include apolymerase and a nucleotide, whereby the sequentially contacting resultsin the primed template nucleic acid being contacted, under the ternarycomplex stabilizing conditions, with nucleotide cognates for first,second and third base type base types in the template; (b) examining theat least two separate mixtures to determine whether a ternary complexformed; and (c) identifying the next correct nucleotide for the primedtemplate nucleic acid molecule, wherein the next correct nucleotide isidentified as a cognate of the first, second or third base type ifternary complex is detected in step (b), and wherein the next correctnucleotide is imputed to be a nucleotide cognate of a fourth base typebased on the absence of a ternary complex in step (b); (d) adding a nextcorrect nucleotide to the primer of the primed template nucleic acidafter step (b), thereby producing an extended primer; and (e) repeatingsteps (a) through (d) for the primed template nucleic acid thatcomprises the extended primer.

Methods for Sequencing with Nucleotides

The present disclosure provides pairwise sequencing compositions andmethods which employ methods for sequencing the plurality of immobilizedconcatemer template molecules and methods for sequencing the pluralityof retained forward extension strands. The sequencing methods comprise:(a) contacting a plurality of sequencing polymerases to (i) a pluralityof nucleic acid template molecules (e.g., a plurality of immobilizedconcatemer template molecules or a plurality of the retained forwardextension strands) and (ii) a plurality of nucleic acid sequencingprimers (e.g., a plurality of the soluble forward sequencing primer or aplurality of the soluble reverse sequencing primers), wherein thecontacting is conducted under a condition suitable to form a pluralityof complexed polymerases each comprising a sequencing polymerase boundto a nucleic acid duplex wherein the nucleic acid duplex comprises anucleic acid template molecule hybridized to a nucleic acid sequencingprimer. In some embodiments, the plurality of sequencing primerscomprise 3′ OH extendible ends. In some embodiments, the solublesequencing primers lack a nucleotide having a scissile moiety whichcomprises uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine. In someembodiments, the plurality of sequencing polymerases compriserecombinant sequencing polymerases. In some embodiments, the pluralityof sequencing polymerases comprise exonuclease-minus sequencingpolymerases.

In some embodiments, the sequencing method further comprises contactingthe (b) contacting the plurality of complexed sequencing polymerase witha plurality of nucleotides under a condition suitable for binding atleast one nucleotide to a complexed sequencing polymerase. In someembodiments, the complexed sequencing polymerase are contacted with theplurality of nucleotides in the presence of at least one catalyticcation that promotes polymerase-catalyzed nucleotide incorporation,where the catalytic cation comprises magnesium and/or manganese. In someembodiments, the plurality of nucleotides comprises at least onenucleotide analog having a chain terminating moiety at the sugar 2′ or3′ position, where the chain terminating moiety is removable or is notremovable. In some embodiments, the plurality of nucleotides comprisesat least one nucleotide that lacks a chain terminating moiety. In someembodiments, the plurality of nucleotides comprises a plurality ofnucleotides labeled with detectable reporter moiety. In someembodiments, the detectable reporter moiety comprises a fluorophore. Insome embodiments, the fluorophore is attached to the nucleotide base. Insome embodiments, the fluorophore is attached to the nucleotide basewith a linker which is cleavable/removable from the base or is notremovable from the base. In some embodiments, at least one of thenucleotides in the plurality is not labeled with a detectable reportermoiety. In some embodiments, a particular detectable reporter moiety(e.g., fluorophore) that is attached to the nucleotide can correspond tothe nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permitdetection and identification of the nucleotide base.

In some embodiments, the sequencing method further comprises (c)incorporating at least one nucleotide into the 3′ end of the extendibleprimer. In some embodiments, the at least one nucleotide binds thecomplexed sequencing polymerase and incorporates into the 3′ end of theextendible primer. In some embodiments, incorporation of the nucleotideinto the 3′ end of the primer in step (c) comprises a primer extensionreaction.

In some embodiments, the sequencing method further comprises (d)detecting the incorporated nucleotide and identifying the nucleo-base ofthe incorporated nucleotide.

In some embodiments, the sequencing method further comprises (e)repeating steps (b), (c) and (d) at least once.

The present disclosure provides pairwise sequencing compositions andmethods which employ methods for sequencing the plurality of immobilizedconcatemer template molecules. The sequencing methods comprise: (a)contacting a plurality of sequencing polymerases to (i) a plurality ofimmobilized concatemer template molecules and (ii) a plurality of thesoluble forward sequencing primers, wherein the contacting is conductedunder a condition suitable to form a plurality of complexed polymeraseseach comprising a sequencing polymerase bound to a nucleic acid duplexwherein the nucleic acid duplex comprises a immobilized concatemertemplate molecule hybridized to a soluble forward sequencing primer; (b)contacting the plurality of complexed sequencing polymerases with aplurality of nucleotides in the presence of at least one catalyticcation that promotes polymerase-catalyzed nucleotide incorporation(e.g., magnesium and/or manganese) and under a condition suitable forbinding at least one nucleotide to a complexed sequencing polymerase,wherein the plurality of nucleotides comprises at least one nucleotideanalog having a removable chain terminating moiety at the sugar 3′position and labeled with a fluorophore; (c) incorporating at least onenucleotide into the 3′ end of the hybridized forward sequencing primersthereby generating a plurality of nascent extended forward sequencingprimers; and (d) detecting the incorporated nucleotide and identifyingthe nucleo-base of the incorporated nucleotide. In some embodiments, thesequencing method further comprises: (e) removing the chain terminatingmoiety from the incorporated nucleotide to generate an extendible 3′ OHgroup on the sugar moiety of the nucleotide while retaining theplurality of complexed polymerases each comprising a sequencingpolymerase bound to a nucleic acid duplex wherein the nucleic acidduplex comprises a immobilized concatemer template molecule hybridizedto a nascent extended forward sequencing primer; (f) contacting theretained complexed polymerases with a plurality of nucleotides in thepresence of at least one catalytic cation that promotespolymerase-catalyzed nucleotide incorporation under a condition suitablefor binding at least one nucleotide to a complexed sequencingpolymerase, wherein the plurality of nucleotides comprises at least onenucleotide analog having a removable chain terminating moiety at thesugar 3′ position and labeled with a fluorophore; (g) incorporating atleast one nucleotide into the 3′ end of the nascent extended forwardsequencing primer; (h) detecting the incorporated nucleotide andidentifying the nucleo-base of the incorporated nucleotide; and (i)repeating steps (e)-(g) at least once. In some embodiments, the removingof step (e) can be conducted using a cleaving reagent. The cleavingreagent can include or lacks one or more compounds that can reducephoto-damage, including antioxidants, triplet state quenchers, singletoxygen quenchers, oxygen scavengers, electron scavengers, anti-fadeformulations.

The present disclosure provides pairwise sequencing compositions andmethods which employ methods for sequencing the plurality of retainedforward extension strands. The sequencing methods comprise: (a)contacting a plurality of sequencing polymerases to (i) a plurality ofthe retained forward extension strands and (ii) a plurality of thesoluble reverse sequencing primers, wherein the contacting is conductedunder a condition suitable to form a plurality of complexed polymeraseseach comprising a sequencing polymerase bound to a nucleic acid duplexwherein the nucleic acid duplex comprises a retained forward extensionstrand hybridized to a soluble reverse sequencing primer; (b) contactingthe plurality of complexed sequencing polymerases with a plurality ofnucleotides in the presence of at least one catalytic cation thatpromotes polymerase-catalyzed nucleotide incorporation (e.g., magnesiumand/or manganese) and under a condition suitable for binding at leastone nucleotide to a complexed sequencing polymerase, wherein theplurality of nucleotides comprises at least one nucleotide analog havinga removable chain terminating moiety at the sugar 3′ position andlabeled with a fluorophore; (c) incorporating at least one nucleotideinto the 3′ end of the hybridized reverse sequencing primers therebygenerating a plurality of nascent extended reverse sequencing primers;and (d) detecting the incorporated nucleotide and identifying thenucleo-base of the incorporated nucleotide. In some embodiments, thesequencing method further comprises: (e) removing the chain terminatingmoiety from the incorporated nucleotide to generate an extendible 3′ OHgroup on the sugar moiety of the nucleotide while retaining theplurality of complexed polymerases each comprising a sequencingpolymerase bound to a nucleic acid duplex wherein the nucleic acidduplex comprises a retained forward extension strand hybridized to anascent extended reverse sequencing primer; (f) contacting the retainedcomplexed polymerases with a plurality of nucleotides in the presence ofat least one catalytic cation that promotes polymerase-catalyzednucleotide incorporation under a condition suitable for binding at leastone nucleotide to a complexed sequencing polymerase, wherein theplurality of nucleotides comprises at least one nucleotide analog havinga removable chain terminating moiety at the sugar 3′ position andlabeled with a fluorophore; (g) incorporating at least one nucleotideinto the 3′ end of the nascent extended reverse sequencing primer; (h)detecting the incorporated nucleotide and identifying the nucleo-base ofthe incorporated nucleotide; and (1) repeating steps (e)-(g) at leastonce. In some embodiments, the removing of step (e) can be conductedusing a cleaving reagent. The cleaving reagent can include or lacks oneor more compounds that can reduce photo-damage, including antioxidants,triplet state quenchers, singlet oxygen quenchers, oxygen scavengers,electron scavengers, anti-fade formulations.

Methods for Sequencing with Multivalent Molecules and Nucleotides

The present disclosure provides pairwise sequencing compositions andmethods which employ methods for sequencing the plurality of immobilizedconcatemer template molecules (or a plurality of immobilizednon-concatemer template molecules) and methods for sequencing theplurality of retained forward extension strands. The sequencing methodsgenerally comprise the steps: (1) conducting a sequencing reaction at aposition on the template molecule using multivalent molecules which bindbut do not incorporate; and (2) conducting a sequencing reaction at thesame position on the template molecule using nucleotides withincorporation; and (3) repeating steps (1) and (2) at the next positionon the template molecule. The nucleotide incorporation of step (2)generates an extended forward sequencing primer strand or an extendedreverse sequencing primer strand. The repeating of steps (1) and (2)generates an extended forward sequencing primer strand or an extendedreverse sequencing primer strand.

The sequencing methods comprise: (a) contacting a plurality of a firstsequencing polymerase to (i) a plurality of nucleic acid templatemolecules (e.g., a plurality of the immobilized concatemer templatemolecules or a plurality of the retained forward extension strands) and(ii) a plurality of soluble nucleic acid sequencing primers (e.g., aplurality of the soluble forward sequencing primer or a plurality of thesoluble reverse sequencing primer), wherein the contacting is conductedunder a condition suitable to form a plurality of first complexedpolymerases each comprising a first sequencing polymerase bound to anucleic acid duplex wherein the nucleic acid duplex comprises thenucleic acid template molecule hybridized to the soluble nucleic acidsequencing primer. In some embodiments, the plurality of sequencingprimers comprise 3′ OH extendible ends. In some embodiments, theplurality of nucleic acid template molecules comprise a plurality of theimmobilized concatemer template molecules and the plurality of nucleicacid sequencing primers comprise a plurality of the soluble forwardsequencing primers. In some embodiments, the plurality of nucleic acidtemplate molecules comprise a plurality of the retained forwardextension strands and the plurality of nucleic acid sequencing primerscomprise a plurality of the soluble reverse sequencing primers. In analternative embodiment, the first and second template molecules areclonally amplified non-concatemer template molecules that are localizedin close proximity to each other. For example, the clonally-amplifiedfirst and second template molecules comprise linear template moleculesthat are generated via bridge amplification and are immobilized to thesame location or feature on a support. The first and secondnon-concatemer template molecules comprise a sequence of interest and atleast one universal sequencing primer binding site. The first and secondsoluble sequencing primers can bind to a sequencing primer binding siteon the first and second non-concatemer template molecules, respectively.

In some embodiments, the sequencing methods further comprise step (b):contacting the plurality of first complexed polymerases with a pluralityof multivalent molecules to form a plurality of multivalent-complexedpolymerases. In some embodiments, individual multivalent molecules inthe plurality of multivalent molecules comprise a core attached tomultiple nucleotide arms and each nucleotide arm is attached to anucleotide (e.g., nucleotide unit) (see FIGS. 104-114 ). In someembodiments, the contacting of step (b) is conducted with a mixture ofany combination of two or more types of multivalent molecules, whereindividual multivalent molecules in the mixture comprise nucleotideunits selected from a group consisting of dATP, dGTP, dCTP, dTTP and/ordUTP. In some embodiments, the contacting of step (b) is conducted undera condition suitable for binding complementary nucleotide units of themultivalent molecules to at least two of the plurality of firstcomplexed polymerases, thereby forming a plurality ofmultivalent-complexed polymerases. The nucleotide unit of a multivalentmolecule can bind the 3′ end of a sequencing primer (or the 3′ end of anascent extended sequencing primer) at a position that is opposite acomplementary nucleotide in the template molecule. In some embodiments,the condition is suitable for inhibiting incorporation of thecomplementary nucleotide units into the sequencing primers (or into thenascent extended sequence primer) of the plurality ofmultivalent-complexed polymerases. In some embodiments, the contactingof step (b) is conducted in the presence of a non-catalytic divalentcation that permits binding of the nucleotide unit to the complexedpolymerase but inhibits polymerase-catalyzed incorporation of thenucleotide unit into the sequencing primer (or into the nascent extendedsequencing primer) of the complexed polymerase, where the non-catalyticdivalent cation comprises strontium and/or barium. In some embodiments,the plurality of multivalent molecules comprise at least one multivalentmolecule having multiple nucleotide arms each attached with a nucleotideanalog (e.g., nucleotide analog unit), where the nucleotide analogincludes a removable chain terminating moiety at the sugar 2′ and/or 3′position. In some embodiments, the plurality of multivalent moleculescomprises at least one multivalent molecule comprising multiplenucleotide arms each attached with a nucleotide unit that lacks a chainterminating moiety. In some embodiments, at least one of the multivalentmolecules in the plurality of multivalent molecules is labeled with adetectable reporter moiety. In individual multivalent molecules, atleast one detectable moiety is attached to the core, or a detectablemoiety is attached to at least one nucleotide unit, or a detectablemoiety is attached to at least one linker. In some embodiments, thedetectable reporter moiety comprises a fluorophore.

In some embodiments, the sequencing methods further comprise step (c):detecting the plurality of multivalent-complexed polymerases. In someembodiments, the detecting includes detecting the multivalent moleculesthat are bound to the complexed polymerases, where the complementarynucleotide units of the multivalent molecules are bound to thesequencing primers but incorporation of the complementary nucleotideunits is inhibited. In some embodiments, the multivalent molecules arelabeled with a detectable reporter moiety to permit detection. In someembodiments, the core is labeled with a detectable reporter moiety. Insome embodiments, at least one linker and/or at least one nucleotideunit of a nucleotide arm is labeled with a detectable reporter moiety.

In some embodiments, the sequencing methods further comprise step (d):identifying the nucleo-base of the complementary nucleotide units thatare bound to the plurality of first complexed polymerases (in theplurality of multivalent-complexes polymerases), thereby determining thesequence of the nucleic acid template. In some embodiments, themultivalent molecules are labeled with a detectable reporter moiety thatcorresponds to the particular nucleotide units attached to thenucleotide arms to permit identification of the complementary nucleotideunits (e.g., nucleotide base adenine, guanine, cytosine, thymine oruracil) that are bound to the plurality of first complexed polymerases.In some embodiments, a given multivalent molecule is labeled with aknown fluorophore to permit differentiation and identification of thenucleo-bases of the given multivalent molecule.

In some embodiments, in the sequencing methods, the binding of theplurality of first complexed polymerases with the plurality ofmultivalent molecules forms at least one avidity complex, the methodcomprising the steps: (1) binding a first soluble sequencing primer(e.g., a soluble forward sequencing primer or a soluble reversesequencing primer), a first sequencing polymerase, and a firstmultivalent molecule to a first portion of a nucleic acid concatemertemplate molecule (e.g., an immobilized concatemer template molecule ora retained forward extension strand) thereby forming a first bindingcomplex (e.g., a first multivalent-complexed polymerase), wherein afirst nucleotide unit of the first multivalent molecule binds to thefirst sequencing polymerase; and (2) binding a second soluble sequencingprimer (e.g., a soluble forward sequencing primer or a soluble reversesequencing primer), a second sequencing polymerase, and the firstmultivalent molecule to a second portion of the same nucleic acidconcatemer template molecule thereby forming a second binding complex(e.g., a second multivalent-complexed polymerase), wherein a secondnucleotide unit of the second multivalent molecule binds to the secondsequencing polymerase, wherein the first and second binding complexeswhich include the same multivalent molecule forms an avidity complex.

In some embodiments, the first sequencing primer comprises a solubleforward sequencing primer and the nucleic acid template moleculecomprises an immobilized concatemer template molecule. In someembodiments, the second sequencing primer comprises a soluble forwardsequencing primer and the nucleic acid template molecule comprises thesame immobilized concatemer template molecule. The first and secondsequencing primers have the same sequence.

In some embodiments, the first sequencing primer comprises a solublereverse sequencing primer and the nucleic acid template moleculecomprises a retained forward extension strand. In some embodiments, thesecond sequencing primer comprises a soluble reverse sequencing primerand the nucleic acid template molecule comprises the same retainedforward extension strand. The first and second sequencing primers havethe same sequence.

In some embodiments, the first sequencing polymerase comprises any wildtype or mutant polymerase described herein. In some embodiments, thesecond sequencing polymerase comprises any wild type or mutantpolymerase described herein. The concatemer template molecule comprisestandem repeat sequences of a sequence of interest and at least oneuniversal sequencing primer binding site. The first and second solublesequencing primers can bind to a sequencing primer binding site alongthe concatemer template molecule.

In some embodiments, the sequencing methods include binding theplurality of first complexed polymerases with the plurality ofmultivalent molecules to form at least one avidity complex, the methodcomprising the steps: (1) contacting a plurality of first sequencingpolymerases and a plurality of second sequencing primers with differentportions of a nucleic acid concatemer template molecule (e.g., animmobilized concatemer template molecule or a retained forward extensionstrand) to form at least first and second complexed polymerases on thesame nucleic acid concatemer template molecule; (2) contacting aplurality of multivalent molecules to the at least first and secondcomplexed polymerases on the same nucleic acid concatemer templatemolecule, under conditions suitable to bind a single multivalentmolecule from the plurality to the first and second complexedpolymerases, wherein at least a first nucleotide unit of the singlemultivalent molecule is bound to the first complexed polymerase whichincludes a first sequencing primer hybridized to a first portion of thenucleic acid concatemer template molecule thereby forming a firstbinding complex (e.g., a first multivalent-complexed polymerase), andwherein at least a second nucleotide unit of the single multivalentmolecule is bound to the second complexed polymerase which includes asecond sequencing primer hybridized to a second portion of the samenucleic acid concatemer template molecule thereby forming a secondbinding complex (e.g., a second multivalent-complexed polymerase),wherein the contacting is conducted under a condition suitable toinhibit polymerase-catalyzed incorporation of the bound first and secondnucleotide units in the first and second binding complexes, and whereinthe first and second binding complexes which are bound to the samemultivalent molecule forms an avidity complex; (3) detecting the firstand second binding complexes on the same nucleic acid concatemertemplate molecule, and (4) identifying the first nucleotide unit in thefirst binding complex thereby determining the sequence of the firstportion of the nucleic acid template molecule, and identifying thesecond nucleotide unit in the second binding complex thereby determiningthe sequence of the second portion of the same nucleic acid templatemolecule.

In some embodiments, the plurality of DNA polymerases comprise any wildtype or mutant polymerase described herein. The concatemer templatemolecule comprises tandem repeat sequences of a sequence of interest andat least one universal sequencing primer binding site. The plurality offirst and second sequencing primers can bind to a sequencing primerbinding site along the concatemer template molecule.

In some embodiments, the plurality of first sequencing primers comprisea plurality of first soluble forward sequencing primers and the nucleicacid concatemer template molecule comprises an immobilized concatemertemplate molecule. In some embodiments, the plurality of secondsequencing primers comprise a plurality of second soluble forwardsequencing primers and the nucleic acid template molecule comprises thesame immobilized concatemer template molecule. The plurality of firstand second sequencing primers have the same sequence.

In some embodiments, the plurality of first sequencing primers comprisea plurality of first soluble reverse sequencing primer and the nucleicacid template molecule comprises a retained forward extension strand. Insome embodiments, the plurality of second sequencing primers comprise aplurality of second soluble reverse sequencing primer and the nucleicacid template molecule comprises the same retained forward extensionstrand. The plurality of first and second sequencing primers have thesame sequence.

In some embodiments, in the sequencing methods, the binding of theplurality of first complexed polymerases with the plurality ofmultivalent molecules forms at least one avidity complex, the methodcomprising the steps: (1) binding a first soluble sequencing primer(e.g., a soluble forward sequencing primer or a soluble reversesequencing primer), a first sequencing polymerase, and a firstmultivalent molecule to a first template molecule (e.g., an immobilizedtemplate molecule or a retained forward extension strand) therebyforming a first binding complex (e.g., a first multivalent-complexedpolymerase), wherein a first nucleotide unit of the first multivalentmolecule binds to the first sequencing polymerase; and (2) binding asecond soluble sequencing primer (e.g., a soluble forward sequencingprimer or a soluble reverse sequencing primer), a second sequencingpolymerase, and the first multivalent molecule to a second templatemolecule thereby forming a second binding complex (e.g., a secondmultivalent-complexed polymerase), wherein a second nucleotide unit ofthe second multivalent molecule binds to the second sequencingpolymerase, wherein the first and second binding complexes which includethe same multivalent molecule forms an avidity complex.

In some embodiments, the first sequencing polymerase comprises any wildtype or mutant polymerase described herein. In some embodiments, thesecond sequencing polymerase comprises any wild type or mutantpolymerase described herein. In some embodiments, the first and secondtemplate molecules are clonally amplified template molecules. In someembodiments, the first and second template molecules are localized inclose proximity to each other. For example, the clonally-amplified firstand second template molecules comprise linear template molecules thatare generated via bridge amplification and are immobilized to the samelocation or feature on a support. The first and second templatemolecules comprise a sequence of interest and at least one universalsequencing primer binding site. The first and second soluble sequencingprimers can bind to a sequencing primer binding site on the first andsecond template molecules, respectively.

In some embodiments, the first soluble sequencing primer comprises asoluble forward sequencing primer and the first template moleculecomprises an immobilized first template molecule. In some embodiments,the second soluble sequencing primer comprises a soluble forwardsequencing primer and the first template molecule comprises the sameimmobilized first template molecule. The first and second solublesequencing primers have the same sequence.

In some embodiments, the first soluble sequencing primer comprises asoluble reverse sequencing primer and the first template moleculecomprises a retained forward extension strand. In some embodiments, thesecond soluble sequencing primer comprises a soluble reverse sequencingprimer and the second template molecule comprises the same retainedforward extension strand. The first and second soluble sequencingprimers have the same sequence.

In some embodiments, the sequencing methods include binding theplurality of first complexed polymerases with the plurality ofmultivalent molecules to form at least one avidity complex, the methodcomprising the steps: (1) contacting a plurality of sequencingpolymerases and a plurality of soluble sequencing primers (whichincludes first and second soluble sequencing primers) with a first andsecond template molecule, respectively (e.g., immobilized first orsecond template molecules, or retained first or second forward extensionstrands) to form at least first and second complexed polymerases on thefirst and second template molecules, respectively; (2) contacting aplurality of multivalent molecules to the at least first and secondcomplexed polymerases, under conditions suitable to bind a singlemultivalent molecule from the plurality to the first and secondcomplexed polymerases, wherein at least a first nucleotide unit of thesingle multivalent molecule is bound to the first complexed polymerasewhich includes a first sequencing primer hybridized to the firsttemplate molecule thereby forming a first binding complex (e.g., a firstmultivalent-complexed polymerase), and wherein at least a secondnucleotide unit of the single multivalent molecule is bound to thesecond complexed polymerase which includes a second sequencing primerhybridized to the second template molecule thereby forming a secondbinding complex (e.g., a second multivalent-complexed polymerase),wherein the contacting is conducted under a condition suitable toinhibit polymerase-catalyzed incorporation of the bound first and secondnucleotide units in the first and second binding complexes, and whereinthe first and second binding complexes which are bound to the samemultivalent molecule forms an avidity complex; (3) detecting the firstand second binding complexes on the first and second template molecules,and (4) identifying the first nucleotide unit in the first bindingcomplex thereby determining the sequence of the first template molecule,and identifying the second nucleotide unit in the second binding complexthereby determining the sequence of the second template molecule.

In some embodiments, the plurality of DNA polymerases comprise any wildtype or mutant polymerase described herein. The first and secondtemplate molecules are clonally amplified template molecules. In someembodiments, the first and second template molecules are localized inclose proximity to each other. For example, the clonally-amplified firstand second template molecules comprise linear template molecules thatare generated via bridge amplification and are immobilized to the samelocation or feature on a support. The first and second templatemolecules comprise a sequence of interest and at least one universalsequencing primer binding site. The first and second soluble primers canbind to a sequencing primer binding site on the first and secondtemplate molecules, respectively.

In some embodiments, the plurality of first sequencing primers comprisea plurality of first soluble forward sequencing primers and the firsttemplate molecule comprises an immobilized first template molecule. Insome embodiments, the plurality of second sequencing primers comprise aplurality of second soluble forward sequencing primers and the secondtemplate molecule comprises an immobilized second template molecule. Theplurality of first and second sequencing primers have the same sequence.

In some embodiments, the plurality of first sequencing primers comprisea plurality of first soluble reverse sequencing primer and the firsttemplate molecule comprises a first retained forward extension strand.In some embodiments, the plurality of second sequencing primers comprisea plurality of second soluble reverse sequencing primer and the secondtemplate molecule comprises a second retained forward extension strand.The plurality of first and second sequencing primers have the samesequence.

In some embodiments, the sequencing methods further comprise step (e):dissociating the plurality of multivalent-complexed polymerases andremoving the plurality of first sequencing polymerases and their boundmultivalent molecules, and retaining the plurality of nucleic acidduplexes.

In some embodiments, the sequencing methods further comprise step (f):contacting the plurality of the retained nucleic acid duplexes of step(e) with a plurality of second sequencing polymerases, wherein thecontacting is conducted under a condition suitable for binding theplurality of second sequencing polymerases to the plurality of theretained nucleic acid duplexes, thereby forming a plurality of secondcomplexed polymerases each comprising a second sequencing polymerasebound to a retained nucleic acid duplex.

In some embodiments, the plurality of first sequencing polymerases ofstep (a) have an amino acid sequence that is 100% identical to the aminoacid sequence as the plurality of the second sequencing polymerases ofstep (f). In some embodiments, the plurality of first sequencingpolymerases of step (a) have an amino acid sequence that differs fromthe amino acid sequence of the plurality of the second sequencingpolymerases of step (f).

In some embodiments, the sequencing methods further comprise step (g):contacting the plurality of second complexed polymerases with aplurality of nucleotides (e.g., free nucleotides), wherein thecontacting is conducted under a condition suitable for bindingcomplementary nucleotides from the plurality of nucleotides to at leasttwo of the second complexed polymerases of step (f) thereby forming aplurality of nucleotide-complexed polymerases. In some embodiments, thecontacting of step (g) is conducted under a condition that is suitablefor promoting incorporation of the bound complementary nucleotides intothe sequencing primers (or the nascent extended sequencing primers) ofthe nucleotide-complexed polymerases, thereby forming a plurality ofnucleotide-complexed polymerases. In some embodiments, incorporating thenucleotide into the 3′ end of the primer in step (g) comprises a primerextension reaction. In some embodiments, the contacting of step (g) isconducted in the presence of a catalytic divalent cation that promotespolymerase-catalyzed nucleotide incorporation. In some embodiments, thecatalytic divalent cation comprises magnesium and/or manganese. In someembodiments, the plurality of nucleotides comprise native nucleotides(e.g., non-analog nucleotides) or nucleotide analogs. In someembodiments, the plurality of nucleotides comprise a 2′ and/or 3′ chainterminating moiety which is removable or is not removable. In someembodiments, the plurality of nucleotides comprises a plurality ofnucleotides labeled with detectable reporter moiety. In someembodiments, the detectable reporter moiety comprises a fluorophore. Insome embodiments, the fluorophore is attached to the nucleotide base. Insome embodiments, the fluorophore is attached to the nucleotide basewith a linker which is cleavable/removable from the base or is notremovable from the base. In some embodiments, at least one of thenucleotides in the plurality is not labeled with a detectable reportermoiety. In some embodiments, a particular detectable reporter moiety(e.g., fluorophore) that is attached to the nucleotide can correspond tothe nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permitdetection and identification of the nucleotide base.

In some embodiments, the sequencing methods further comprise step (h):detecting the complementary nucleotides which are incorporated into thesequencing primers (or the nascent extended sequencing primers) of thenucleotide-complexed polymerases. In some embodiments, the plurality ofnucleotides are labeled with a detectable reporter moiety to permitdetection. In some embodiments, the detecting step (h) is omitted.

In some embodiments, the sequencing methods further comprise step (i):identifying the nucleo-bases of the complementary nucleotides which areincorporated into the sequencing primers (or the nascent extendedsequencing primers) of the nucleotide-complexed polymerases. In someembodiments, the identification of the incorporated complementarynucleotides in step (i) can be used to confirm the identity of thecomplementary nucleotide units of the multivalent molecules that werebound to the plurality of first complexed polymerases in step (d). Insome embodiments, the identifying of step (i) can be used to determinethe sequence of the nucleic acid template molecules. In someembodiments, the identifying step (i) is omitted.

In some embodiments, the sequencing methods further comprises step (j):removing the chain terminating moiety from the incorporated nucleotidewhen step (g) is conducted by contacting the plurality of secondcomplexed polymerases with a plurality of nucleotides that comprise atleast one nucleotide having a 2′ and/or 3′ chain terminating moiety. Insome embodiments, the removing of step (j) can be conducted using acleaving reagent. The cleaving reagent can include or lacks one or morecompounds that can reduce photo-damage, including antioxidants, tripletstate quenchers, singlet oxygen quenchers, oxygen scavengers, electronscavengers, anti-fade formulations.

In some embodiments, the sequencing methods further comprise step (k):repeating steps (a)-(j) at least once, by using the same nucleic acidsequencing primers of step (a). In some embodiments, the sequence of thenucleic acid template molecules can be determined by detecting andidentifying the multivalent molecules that bind the sequencingpolymerases but do not incorporate into the 3′ end of the primer atsteps (c) and (d). In some embodiments, the sequence of the nucleic acidtemplate molecule can be determined (or confirmed) by detecting andidentifying the nucleotide that incorporates into the 3′ end of theprimer at steps (h) and (i).

In an alternative sequencing method, the steps for forming a pluralityof complexed polymerases further comprise step (b2): contacting theplurality of complexed polymerases of step (a) with a plurality ofnucleotides under a condition suitable for binding a complementarynucleotide from the plurality of nucleotides to a complexed polymerasefrom the plurality of complexed polymerases, thereby forming anucleotide-complexed polymerase. In some embodiments, the contacting ofstep (b2) is conducted under a condition that is suitable for promotingnucleotide binding but inhibiting incorporation of the boundcomplementary nucleotides to the 3′ end of the primers of thenucleotide-complexed polymerases. In some embodiments, the contacting ofstep (b2) is conducted in the presence of at least one cation selectedfrom a group consisting of strontium, barium, sodium, magnesium,potassium, manganese, calcium, lithium, nickel and cobalt. The pluralityof complexed polymerases can be contacted sequentially with at least twoseparate mixtures where each mixture comprises an engineered polymeraseand a nucleotide. The contacting is conducted under conditions suitablefor forming stable ternary complexes with cognates for first, second andthird base type base types in the template. The method further comprisesstep (c3) examining the at least two separate mixtures to determine if aternary complex formed. The method further comprises step (d3)identifying the next correct nucleotide for the primed template nucleicacid molecule, wherein the next correct nucleotide is identified as acognate of the first, second or third base type if ternary complex isdetected in step (c3), and wherein the next correct nucleotide isimputed to be a nucleotide cognate of a fourth base type based on theabsence of a ternary complex in step (c3). The method further comprisesstep (e3) adding a next correct nucleotide to the primer of the primedtemplate nucleic acid after step (c3), thereby producing an extendedprimer; and step (f3) repeating steps (a) through (e3) for the primedtemplate nucleic acid that comprises the extended primer.

Nucleotides

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides comprise a base,sugar and at least one phosphate group. In some embodiments, at leastone nucleotide in the plurality comprises an aromatic base, a fivecarbon sugar (e.g., ribose or deoxyribose), and one or more phosphategroups (e.g., 1-10 phosphate groups). The plurality of nucleotides cancomprise at least one type of nucleotide selected from a groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP. The plurality ofnucleotides can comprise at a mixture of any combination of two or moretypes of nucleotides selected from a group consisting of dATP, dGTP,dCTP, dTTP and/or dUTP. In some embodiments, at least one nucleotide inthe plurality is not a nucleotide analog. In some embodiments, at leastone nucleotide in the plurality comprises a nucleotide analog.

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides comprise a chainof one, two or three phosphorus atoms where the chain is typicallyattached to the 5′ carbon of the sugar moiety via an ester orphosphoramide linkage. In some embodiments, at least one nucleotide inthe plurality is an analog having a phosphorus chain in which thephosphorus atoms are linked together with intervening O, S, NH,methylene or ethylene. In some embodiments, the phosphorus atoms in thechain include substituted side groups including O, S or BH₃. In someembodiments, the chain includes phosphate groups substituted withanalogs including phosphoramidate, phosphorothioate, phosphordithioate,and O-methylphosphoroamidite groups.

In some embodiments, in any of the sequencing methods described herein,the plurality of nucleotides comprises a plurality of nucleotideslabeled with detectable reporter moiety. The detectable reporter moietycomprises a fluorophore. In some embodiments, the fluorophore isattached to the nucleotide base. In some embodiments, the fluorophore isattached to the nucleotide base with a linker which iscleavable/removable from the base. In some embodiments, at least one ofthe nucleotides in the plurality is not labeled with a detectablereporter moiety. In some embodiments, a particular detectable reportermoiety (e.g., fluorophore) that is attached to the nucleotide cancorrespond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP)to permit detection and identification of the nucleotide base. In someembodiments, the method further comprises detecting the at least oneincorporated nucleotide at step (c) and/or (d). In some embodiments, themethod further comprises identifying the at least one incorporatednucleotide at step (c) and/or (d). In some embodiments, the sequence ofthe nucleic acid template molecule can be determined by detecting andidentifying the nucleotide (nucleo-base) that binds the sequencingpolymerase, thereby determining the sequence of the nucleic acidtemplate. In some embodiments, the sequence of the nucleic acid templatemolecule can be determined by detecting and identifying the nucleotide(nucleo-base) that incorporates into the 3′ end of the primer, therebydetermining the sequence of the nucleic acid template. In someembodiments, detection and identification of the fluorescently-labelednucleotides can be achieved by imaging the support upon which thesequencing reaction is conducted.

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides comprises aterminator nucleotide analog having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position. In some embodiments, the chainterminating moiety can inhibit polymerase-catalyzed incorporation of asubsequent nucleotide unit or free nucleotide in a nascent strand duringa primer extension reaction. In some embodiments, the chain terminatingmoiety is attached to the 3′ sugar hydroxyl position where the sugarcomprises a ribose or deoxyribose sugar moiety. In some embodiments, thechain terminating moiety is removable/cleavable from the 3′ sugarhydroxyl position to generate a nucleotide having a 3′ OH sugar groupwhich is extendible with a subsequent nucleotide in apolymerase-catalyzed nucleotide incorporation reaction. In someembodiments, the chain terminating moiety comprises an alkyl group,alkenyl group, alkynyl group, allyl group, aryl group, benzyl group,azide group, amine group, amide group, keto group, isocyanate group,phosphate group, thio group, disulfide group, carbonate group, ureagroup, or silyl group. In some embodiments, the chain terminating moietyis cleavable/removable from the nucleotide, for example by reacting thechain terminating moiety with a chemical agent, pH change, light orheat. In some embodiments, the chain terminating moieties alkyl,alkenyl, alkynyl and allyl are cleavable withtetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) with piperidine, orwith 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In someembodiments, the chain terminating moieties aryl and benzyl arecleavable with H2 Pd/C. In some embodiments, the chain terminatingmoieties amine, amide, keto, isocyanate, phosphate, thio, disulfide arecleavable with phosphine or with a thiol group includingbeta-mercaptoethanol or dithiothritol (DTT). In some embodiments, thechain terminating moiety carbonate is cleavable with potassium carbonate(K₂CO₃) in MeOH, with triethylamine in pyridine, or with Zn in aceticacid (AcOH). In some embodiments, the chain terminating moieties ureaand silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF,with ammonium fluoride, or with triethylamine trihydrofluoride.

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides comprises aterminator nucleotide analog having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ position, at the sugar 3′ position, orat the sugar 2′ and 3′ position. In some embodiments, the chainterminating moiety comprises an azide, azido or azidomethyl group. Insome embodiments, the chain terminating moiety comprises a 3′-O-azido or3′-O-azidomethyl group. In some embodiments, the chain terminatingmoieties azide, azido and azidomethyl group are cleavable/removable witha phosphine compound. In some embodiments, the phosphine compoundcomprises a derivatized tri-alkyl phosphine moiety or a derivatizedtri-aryl phosphine moiety. In some embodiments, the phosphine compoundcomprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenylphosphine (BS-TPP) or Tris(hydroxyproyl)phosphine (THPP) orTris(hydroxymethyl)phosphine (THMP). In some embodiments, the cleavingagent comprises 4-dimethylaminopyridine (4-DMAP).

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides comprises aterminator nucleotide analog having a chain terminating moiety (e.g.,blocking moiety) at the sugar 2′ and/or 3′ position, where theincorporated chain terminating nucleotide is removed/cleaved bycontacting the incorporated chain terminating nucleotide with a cleavingreagent. The cleaving reagent can include or lacks one or more compoundsthat can reduce photo-damage, including antioxidants, triplet statequenchers, singlet oxygen quenchers, oxygen scavengers, electronscavengers, anti-fade formulations.

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides comprises achain terminating moiety which is selected from a group consisting of3′-deoxy nucleotides, 2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido,3′-azidomethyl, 3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl,3′-O-fluoroalkyl, 3′-fluoromethyl, 3′-difluoromethyl,3′-trifluoromethyl, 3′-sulfonyl, 3′-malonyl, 3′-amino, 3′-O-amino,3′-sulfhydral, 3′-aminomethyl, 3′-ethyl, 3′butyl, 3′-tert butyl,3′-Fluorenylmethyloxycarbonyl, 3′ tert-Butyloxycarbonyl, 3′-O-alkylhydroxylamino group, 3′-phosphorothioate, and 3-O-benzyl, or derivativesthereof.

In some embodiments, in any of the sequencing methods described herein,at least one nucleotide in the plurality of nucleotides is labeled withdetectable reporter moiety. The detectable reporter moiety comprises afluorophore. In some embodiments, the fluorophore is attached to thenucleotide base. In some embodiments, the fluorophore is attached to thenucleotide base with a linker which is cleavable/removable from thebase. In some embodiments, at least one of the nucleotides in theplurality is not labeled with a detectable reporter moiety. In someembodiments, a particular detectable reporter moiety (e.g., fluorophore)that is attached to the nucleotide can correspond to the nucleotide base(e.g., dATP, dGTP, dCTP, dTTP or dUTP) to permit detection andidentification of the nucleotide base.

In some embodiments, in any of the sequencing methods described herein,the cleavable linker on the base comprises a cleavable moiety comprisingan alkyl group, alkenyl group, alkynyl group, allyl group, aryl group,benzyl group, azide group, amine group, amide group, keto group,isocyanate group, phosphate group, thio group, disulfide group,carbonate group, urea group, or silyl group. In some embodiments, thecleavable linker on the base is cleavable/removable from the base byreacting the cleavable moiety with a chemical agent, pH change, light orheat. In some embodiments, the cleavable moieties alkyl, alkenyl,alkynyl and allyl are cleavable withtetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) with piperidine, orwith 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In someembodiments, the cleavable moieties aryl and benzyl are cleavable withH2 Pd/C. In some embodiments, the cleavable moieties amine, amide, keto,isocyanate, phosphate, thio, disulfide are cleavable with phosphine orwith a thiol group including beta-mercaptoethanol or dithiothritol(DTT). In some embodiments, the cleavable moiety carbonate is cleavablewith potassium carbonate (K₂CO₃) in MeOH, with triethylamine inpyridine, or with Zn in acetic acid (AcOH). In some embodiments, thecleavable moieties urea and silyl are cleavable with tetrabutylammoniumfluoride, pyridine-HF, with ammonium fluoride, or with triethylaminetrihydrofluoride.

In some embodiments, in any of the sequencing methods described herein,the cleavable linker on the base comprises cleavable moiety including anazide, azido or azidomethyl group. In some embodiments, the cleavablemoieties azide, azido and azidomethyl group are cleavable/removable witha phosphine compound. In some embodiments, the phosphine compoundcomprises a derivatized tri-alkyl phosphine moiety or a derivatizedtri-aryl phosphine moiety. In some embodiments, the phosphine compoundcomprises Tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenylphosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In someembodiments, the cleaving agent comprises 4-dimethylaminopyridine(4-DMAP).

In some embodiments, in any of the sequencing methods described herein,the chain terminating moiety (e.g., at the sugar 2′ and/or sugar 3′position) and the cleavable linker on the base have the same ordifferent cleavable moieties. In some embodiments, the chain terminatingmoiety (e.g., at the sugar 2′ and/or sugar 3′ position) and thedetectable reporter moiety linked to the base are chemicallycleavable/removable with the same chemical agent. In some embodiments,the chain terminating moiety (e.g., at the sugar 2′ and/or sugar 3′position) and the detectable reporter moiety linked to the base arechemically cleavable/removable with different chemical agents.

Multivalent Molecules

The present disclosure provides compositions and methods for pairwisesequencing, including methods for sequencing the plurality ofimmobilized concatemer template molecules and methods for sequencing theplurality of retained forward extension strands, where the sequencingmethods employ multivalent molecules. In some embodiments, in thesequencing methods, at least one multivalent molecule in the pluralityof multivalent molecules of step (b) comprises: (1) a core; and (2) aplurality of nucleotide arms which comprise (i) a core attachmentmoiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv)a nucleotide unit, wherein the core is attached to the plurality ofnucleotide arms, wherein the spacer is attached to the linker, whereinthe linker is attached to the nucleotide unit. In some embodiments, thenucleotide unit comprises a base, sugar and at least one phosphategroup, and the linker is attached to the nucleotide unit through thebase. In some embodiments, the linker comprises an aliphatic chain or anoligo ethylene glycol chain where both linker chains having 2-6subunits. In some embodiments, the linker also includes an aromaticmoiety. Exemplary multivalent molecules are shown in FIGS. 104-107 .Exemplary nucleotide arms are shown in FIGS. 108 and 114 . An exemplaryspacer is shown in FIG. 109 (top), and exemplary linkers are shown inFIGS. 109 (bottom) and 110-113.

In some embodiments, in the sequencing methods, individual multivalentmolecules in the plurality of multivalent molecules of step (b) comprisea core attached to multiple nucleotide arms, and wherein the multiplenucleotide arms have the same type of nucleotide unit which is selectedfrom a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.Alternatively, the multivalent molecule comprises a core attached tomultiple nucleotide arms where different nucleotide arms are attached toa different type of nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP ordUTP) and the multivalent molecule comprises any combination of two ormore different nucleotide units.

In some embodiments, in the sequencing methods, the nucleotide unit ofthe at least one multivalent molecule of step (b) comprises an aromaticbase, a five carbon sugar (e.g., ribose or deoxyribose), and one or morephosphate groups (e.g., 1-10 phosphate groups). The plurality ofmultivalent molecules can comprise one type of multivalent moleculehaving one type of nucleotide unit selected from a group consisting ofdATP, dGTP, dCTP, dTTP and dUTP. The plurality of multivalent moleculescan comprise at a mixture of any combination of two or more types ofmultivalent molecules, where individual multivalent molecules in themixture comprise nucleotide units selected from a group consisting ofdATP, dGTP, dCTP, dTTP and/or dUTP.

In some embodiments, in the sequencing methods, at least one multivalentmolecule in the plurality of multivalent molecules of step (b) comprisea nucleotide unit having a chain of one, two or three phosphorus atomswhere the chain is typically attached to the 5′ carbon of the sugarmoiety via an ester or phosphoramide linkage. In some embodiments, atleast one nucleotide unit is a nucleotide analog having a phosphoruschain in which the phosphorus atoms are linked together with interveningO, S, NH, methylene or ethylene. In some embodiments, the phosphorusatoms in the chain include substituted side groups including O, S orBH₃. In some embodiments, the chain includes phosphate groupssubstituted with analogs including phosphoramidate, phosphorothioate,phosphordithioate, and O-methylphosphoroamidite groups.

In some embodiments, in the sequencing methods, individual multivalentmolecules in the plurality of multivalent molecule of step (b) comprisea core attached to multiple nucleotide arms, and wherein individualnucleotide arms comprise a nucleotide unit having a chain terminatingmoiety (e.g., blocking moiety) at the sugar 2′ position, at the sugar 3′position, or at the sugar 2′ and 3′ position. An exemplary nucleotidearm is shown in FIG. 108 , and exemplary multivalent molecules are shownin FIGS. 104-107 .

In some embodiments, in the sequencing methods, at least one multivalentmolecule in the plurality of multivalent molecules of step (b) comprisesa nucleotide unit comprising a terminator nucleotide analog having achain terminating moiety (e.g., blocking moiety) at the sugar 2′position, at the sugar 3′ position, or at the sugar 2′ and 3′ position.In some embodiments, the chain terminating moiety can inhibitpolymerase-catalyzed incorporation of a subsequent nucleotide unit orfree nucleotide in a nascent strand during a primer extension reaction.In some embodiments, the chain terminating moiety is attached to the 3′sugar hydroxyl position where the sugar comprises a ribose ordeoxyribose sugar moiety. In some embodiments, the chain terminatingmoiety is removable/cleavable from the 3′ sugar hydroxyl position togenerate a nucleotide having a 3′OH sugar group which is extendible witha subsequent nucleotide in a polymerase-catalyzed nucleotideincorporation reaction. In some embodiments, the chain terminatingmoiety comprises an alkyl group, alkenyl group, alkynyl group, allylgroup, aryl group, benzyl group, azide group, amine group, amide group,keto group, isocyanate group, phosphate group, thio group, disulfidegroup, carbonate group, urea group, or silyl group. In some embodiments,the chain terminating moiety is cleavable/removable from the nucleotideunit, for example by reacting the chain terminating moiety with achemical agent, pH change, light or heat. In some embodiments, the chainterminating moieties alkyl, alkenyl, alkynyl and allyl are cleavablewith tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) withpiperidine, or with 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). Insome embodiments, the chain terminating moieties aryl and benzyl arecleavable with H2 Pd/C. In some embodiments, the chain terminatingmoieties amine, amide, keto, isocyanate, phosphate, thio, disulfide arecleavable with phosphine or with a thiol group includingbeta-mercaptoethanol or dithiothritol (DTT). In some embodiments, thechain terminating moiety carbonate is cleavable with potassium carbonate(K₂CO₃) in MeOH, with triethylamine in pyridine, or with Zn in aceticacid (AcOH). In some embodiments, the chain terminating moieties ureaand silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF,with ammonium fluoride, or with triethylamine trihydrofluoride.

In some embodiments, in the sequencing methods, at least one multivalentmolecule in the plurality of multivalent molecules of step (b) comprisesa nucleotide unit comprising a terminator nucleotide analog having achain terminating moiety (e.g., blocking moiety) at the sugar 2′position, at the sugar 3′ position, or at the sugar 2′ and 3′ position.In some embodiments, the chain terminating moiety comprises an azide,azido or azidomethyl group. In some embodiments, the chain terminatingmoiety comprises a 3′-O-azido or 3′-O-azidomethyl group. In someembodiments, the chain terminating moieties azide, azido and azidomethylgroup are cleavable/removable with a phosphine compound. In someembodiments, the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-aryl phosphine moiety. In someembodiments, the phosphine compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP) or Tri(hydroxyproyl)phosphine (THPP). In some embodiments, thecleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

In some embodiments, in the sequencing methods, at least one multivalentmolecule in the plurality of multivalent molecules of step (b) comprisesa nucleotide unit comprising a chain terminating moiety which isselected from a group consisting of 3′-deoxy nucleotides,2′,3′-dideoxynucleotides, 3′-methyl, 3′-azido, 3′-azidomethyl,3′-O-azidoalkyl, 3′-O-ethynyl, 3′-O-aminoalkyl, 3′-O-fluoroalkyl,3′-fluoromethyl, 3′-difluoromethyl, 3′-trifluoromethyl, 3′-sulfonyl,3′-malonyl, 3′-amino, 3′-O-amino, 3′-sulfhydral, 3′-aminomethyl,3′-ethyl, 3′butyl, 3 ‘-tert butyl, 3’-Fluorenylmethyloxycarbonyl, 3′tert-Butyloxycarbonyl, 3′-O-alkyl hydroxylamino group,3′-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

In some embodiments, in the sequencing methods, at least one multivalentmolecule in the plurality of multivalent molecules of step (b) comprisesa core attached to multiple nucleotide arms, wherein the core is labeledwith detectable reporter moiety, or at least one nucleotide unit islabeled with a detectable reporter moiety, or at least one linker islabeled with a detectable reporter moiety. In some embodiments, thedetectable reporter moiety comprises a fluorophore. In some embodiments,a particular detectable reporter moiety (e.g., fluorophore) that isattached to the multivalent molecule can correspond to the base (e.g.,dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide unit to permitdetection and identification of the nucleotide base.

In some embodiments, in the sequencing methods, at least one nucleotidearm of a multivalent molecule in the plurality of multivalent moleculesof step (b) has a nucleotide unit that is attached to a detectablereporter moiety. In some embodiments, the detectable reporter moiety isattached to the nucleotide base. In some embodiments, the detectablereporter moiety comprises a fluorophore. In some embodiments, aparticular detectable reporter moiety (e.g., fluorophore) that isattached to the multivalent molecule can correspond to the base (e.g.,dATP, dGTP, dCTP, dTTP or dUTP) of the nucleotide unit to permitdetection and identification of the nucleotide base.

In some embodiments in the sequencing methods, the core of themultivalent molecule can be labeled with a detectable reporter moiety(e.g., fluorophore) in a manner that permits distinction betweendifferent multivalent molecules carrying a different type of nucleotideunit. For example, the core of a first multivalent molecule is labeledwith a first fluorophore, where the first multivalent molecule comprisesmultiple nucleotide-arms with dGTP nucleotide units. The core of asecond multivalent molecule is labeled with a second fluorophore (whichdiffers from the first fluorophore), where the second multivalentmolecule comprises multiple nucleotide-arms with dATP nucleotide units.The binding and incorporating events of the nucleotide unit can bedetected, and the specific base of the nucleotide unit (as part of themultivalent molecule) can be identified based on detection andidentification of the detectable reporter moiety on the first and secondcore.

In some embodiments in the sequencing methods, at least one linker of anucleotide-arm of a multivalent molecule can be labeled with adetectable reporter moiety (e.g., fluorophore) in a manner that permitsdistinction between different multivalent molecules carrying a differenttype of nucleotide unit. For example, at least one linker of a firstmultivalent molecule is labeled with a first fluorophore, where thefirst multivalent molecule comprises multiple nucleotide-arms with dGTPnucleotide units. At least one linker of a second multivalent moleculeis labeled with a second fluorophore (which differs from the firstfluorophore), where the second multivalent molecule comprises multiplenucleotide-arms with dATP nucleotide units. The binding andincorporating events of the nucleotide units can be detected, and thespecific base of the nucleotide unit (as part of the multivalentmolecule) can be identified based on detection and identification of thedetectable reporter moiety on the first and second linkers.

In some embodiments in the sequencing methods, at least one nucleotideunit (e.g., nucleo-base) of a nucleotide-arm of a multivalent moleculecan be labeled with a detectable reporter moiety (e.g., fluorophore) ina manner that permits distinction between different multivalentmolecules carrying a different type of nucleotide unit. For example, atleast one nucleotide unit of a first multivalent molecule is labeledwith a first fluorophore, where the first multivalent molecule comprisesmultiple nucleotide-arms with dGTP nucleotide units. At least onenucleotide unit of a second multivalent molecule is labeled with asecond fluorophore (which differs from the first fluorophore), where thesecond multivalent molecule comprises multiple nucleotide-arms with dATPnucleotide units. The binding and incorporating events of the nucleotideunits can be detected, and the specific base of the nucleotide unit (aspart of the multivalent molecule) can be identified based on detectionand identification of the detectable reporter moiety on the first andsecond nucleotide units.

In some embodiments, in the sequencing methods, the core of amultivalent molecule of step (b) comprises a streptavidin-type oravidin-type moiety and the core attachment moiety comprises biotin. Insome embodiments, the core comprises an streptavidin-type or avidin-typemoiety which includes a streptavidin or avidin protein, as well as anyderivatives, analogs and other non-native forms of avidin that can bindto at least one biotin moiety. Other forms of streptavidin or avidinmoieties include native and recombinant avidin and streptavidin as wellas derivatized molecules, e.g. non-glycosylated avidin and truncatedstreptavidins. For example, avidin moiety includes de-glycosylated formsof avidin, bacterial streptavidin produced by Streptomyces (e.g.,Streptomyces avidinii), as well as derivatized forms, for example,N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, andthe commercially-available products ExtrAvidin™, Captavidin™,Neutravidin™, and Neutralite Avidin™.

In some embodiments, in the sequencing methods, the core of amultivalent molecule of step (b) comprises an streptavidin-type oravidin-type moiety which is labeled with at least one detectablereporter moiety, such as for example a fluorophore. Thestreptavidin-type or avidin-type core can be labeled with 1, 2, 3, 4 ormore detectable moieties. The streptavidin-type or avidin-type core canbe labeled with one or more of the same type of detectable reportermoiety. In some embodiments, a particular detectable reporter moiety(e.g., fluorophore) that is attached to the streptavidin-type oravidin-type core can correspond to the base (e.g., dATP, dGTP, dCTP,dTTP or dUTP) of the nucleotide units to permit detection andidentification of the multivalent molecule.

It is postulated that an increase in binding of a nucleotide to anenzyme (e.g., polymerase) or an enzyme complex can be effected byincreasing the effective concentration of the nucleotide. The increasecan be achieved by increasing the concentration of the nucleotide infree solution, or by increasing the amount of the nucleotide inproximity to the relevant binding site. The increase can also beachieved by physically restricting a number of nucleotides into alimited volume thus resulting in a local increase in concentration, andsuch as structure may thus bind to the binding site with a higherapparent avidity than would be observed with unconjugated, untethered,or otherwise unrestricted individual nucleotides. One exemplary means ofeffecting such restriction is by providing a multivalent molecule inwhich multiple nucleotides are bound to a particle (or core) such as apolymer, a branched polymer, a dendrimer, a micelle, a liposome, amicroparticle, a nanoparticle, a quantum dot, or other suitable particleknown in the art.

The multivalent molecule can increase the effective concentration of thenucleotide in free solution, or can increase the amount of nucleotide inproximity to the nucleotide binding site or the nucleotide incorporationsite of the polymerase. The increase in effective concentration can alsobe achieved by physically restricting the number of nucleotides into alimited volume, thus increasing the local nucleotide concentration. Themultivalent molecule can bind to the polymerase with an increasedapparent avidity compared to an unconjugated nucleotide. When thenucleotide unit is complementary to the interrogation position in thetemplate molecule, the multivalent molecule forms a binding complex withthe polymerase and the template molecule, and the binding complexexhibits increased stability and longer persistence time than a bindingcomplex formed using a single unconjugated or untethered nucleotide(e.g., a free nucleotide). When bound to a polymerase, the multivalentmolecule can increase the local concentration of nucleotides increasesmany-fold, which in turn enhances signal intensity. When bound to apolymerase, the multivalent molecule can exhibit longer persistence timewhich can shorten the imaging a fluorescent signal for the detecting andidentifying steps. When bound to a polymerase, the multivalent moleculeforms a stable complex with the polymerase. The stable complex canwithstand washing steps, so that the signal intensity remains highthroughout the imaging and washing steps. The stable complex can bedissociated (e.g., by changing buffer composition) so that the primedtarget nucleic acid can undergo extension in a subsequent reaction witha free nucleotide.

The multivalent molecule can be used to localize detectable signals toactive regions of biochemical interactions, such as sites ofprotein-nucleic acid interactions, nucleic acid hybridization reactions,or enzymatic reactions, such as polymerase reactions. For example, themultivalent molecules described herein can be utilized to identify sitesof base binding to a nucleic acid template molecule or baseincorporation in elongating nucleic acid chains duringpolymerase-catalyzed reactions and to provide base discrimination forsequencing and array based applications. The increased binding betweenthe nucleic acid template molecule and the nucleotide unit of themultivalent binding composition, when the nucleotide is complementary tothe target nucleic acid, provides enhanced signal that greatly improvebase call accuracy and shortens imaging time.

In addition, the use of multivalent molecules allows sequencing signalsfrom a given template molecule to originate within polony regionscontaining multiple copies of the template sequence. Sequencing methodsthat include multiple copies of a target sequence (e.g., concatemermolecules) have the advantage that signals can be amplified due to thepresence of multiple simultaneous sequencing reactions within thedefined region, each providing its own signal. The presence of multiplesignals within a defined area also reduces the impact of any singleskipped cycle, due to the fact that the signal from a large number ofcorrect base calls can overwhelm the signal from a smaller number ofskipped or incorrect base calls, therefore providing methods forreducing phasing errors and/or to improve read length in sequencingreactions.

The multivalent molecules and their use disclosed herein lead to one ormore of: (i) stronger signal for better base-calling accuracy comparedto conventional nucleic acid amplification and sequencing methodologies;ii) allow greater discrimination of sequence-specific signal frombackground signals; (iii) reduced requirements for the amount ofstarting material necessary, (iv) increased sequencing rate andshortened sequencing time; (v) reducing phasing errors, and (vi)improving read length in sequencing reactions.

One of ordinary skill would recognize that in a series of iterativesequencing reactions, occasionally one or more sites will fail toincorporate a nucleotide during a given cycle, thus leading one or moresites to be unsynchronized with the bulk of the elongating nucleic acidchains. Under a condition in which sequencing signals are derived fromreactions occurring on single copies of a target nucleic acid, thesefailures to incorporate will yield discrete errors in the outputsequence. Use of the multivalent molecules for sequencing can reducethis type of error in sequencing reactions. For example, the use ofmultivalent substrates that are capable of binding to apolymerase-template-primer complex, or capable of incorporation into theelongating strand, by providing increased probabilities of rebindingupon premature dissociation of a ternary polymerase complex, can reducethe frequency of “skipped” cycles in which a base is not incorporated.Thus, in some embodiments, the present disclosure contemplates the useof multivalent molecules as disclosed herein comprising a nucleotidehaving a free, or reversibly modified, 5′ phosphate, diphosphate, ortriphosphate moiety, and wherein the nucleotide is connected to theparticle or polymer as disclosed herein, through a labile or cleavablelinkage. In some embodiments, the present disclosure contemplates areduction in the intrinsic error rate due to skipped incorporations as aresult of the use of the multivalent substrates disclosed herein.

When each sequencing cycle proceeds perfectly, each reaction within thedefined region will provide an identical signal. However, as notedelsewhere herein, in a series of iterative sequencing reactions,occasionally one or more sites will fail to incorporate a nucleotideduring a given cycle, thus leading one or more sites to beunsynchronized with the bulk of the elongating nucleic acid chains. Thisissue, referred to as “phasing,” leads to degradation of the sequencingsignal as the signal is contaminated with spurious signals from siteshaving skipped one or more cycles. This, in turn, creates the potentialfor errors in base identification. The progressive accumulation ofskipped cycles through multiple cycles also reduces the effective readlength, due to progressive degradation of the sequencing signal witheach cycle. It is a further object of this disclosure to provide methodsfor reducing phasing errors and/or to improve read length in sequencingreactions.

Sequencing methods that employ multivalent molecules can result inreduced error rate as indicated by reduction in the misidentification ofbases, the reporting of nonexistent bases, or the failure to reportcorrect bases. In some embodiments, the reduction in the error rate canin be a reduction of about 5%, 10%, 15%, 20% 25%, 50%, 75%, 100%, 150%,200%, or more compared to the error rate observed using monovalentligands, including free nucleotides, labeled free nucleotides, proteinor peptide bound nucleotides, or labeled protein or peptide boundnucleotides.

Sequencing methods that employ multivalent molecules can result inincreased average read length of about 5%, 10%, 15%, 20% 25%, 50%, 75%,100%, 150%, 200%, 300%, or more compared to the average read lengthobserved using monovalent ligands, including free nucleotides, labeledfree nucleotides, protein or peptide bound nucleotides, or labeledprotein or peptide bound nucleotides.

Sequencing methods that employ multivalent molecules can result inincreased in average read length of about 10, 20, 25, 30, 50, 75, 100,125, 150, 200, 250, 300, 350, 400, 500 nucleotides, or more compared tothe average read length observed using monovalent ligands, includingfree nucleotides, labeled free nucleotides, protein or peptide boundnucleotides, or labeled protein or peptide bound nucleotides.

The use of the multivalent molecules for sequencing may effectivelyshortens the sequencing time. The sequencing reaction cycle comprisingthe contacting, detecting, and incorporating steps is performed in atotal time ranging from about 5 minutes to about 60 minutes. In someembodiments, the sequencing reaction cycle is performed in at least 5minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes,at least 40 minutes, at least 50 minutes, or at least 60 minutes. Insome embodiments, the sequencing reaction cycle is performed in at most60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes,at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome embodiments the sequencing reaction cycle may be performed in atotal time ranging from about 10 minutes to about 30 minutes. Those ofskill in the art will recognize that the sequencing cycle time may haveany value within this range, e.g., about 16 minutes.

The use of the multivalent molecules for sequencing may provide improvedaccuracy base readout. The disclosed compositions and methods fornucleic acid sequencing will provide an average Q-score for base-callingaccuracy over a sequencing run that ranges from about 20 to about 50. Insome embodiments, the average Q-score is at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, or at least 50. Thoseof skill in the art will recognize that the average Q-score may have anyvalue within this range, e.g., about 32. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 30 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified. In some embodiments, the disclosed compositions and methodsfor nucleic acid sequencing will provide a Q-score of greater than 35for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 40 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified. In some embodiments, the disclosed compositions and methodsfor nucleic acid sequencing will provide a Q-score of greater than 45for at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% of theterminal (or N+1) nucleotides identified. In some embodiments, thedisclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

In some embodiments, a multivalent molecule comprises a core comprisinga branched polymer; a dendrimer; a cross linked polymer particle such asan agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate,methyl methacrylate particle; a glass particle; a ceramic particle; ametal particle; a quantum dot; a liposome; an emulsion particle, or anyother particle (e.g., nanoparticles, microparticles, or the like) knownin the art.

The particle or core can also have a binding moiety. In someembodiments, particles or cores may self-associate without the use of aseparate interaction moiety. In some embodiments, particles or cores mayself-associate due to buffer conditions or salt conditions, e.g., as inthe case of calcium-mediated interactions of hydroxyapatite particles,lipid or polymer mediated interactions of micelles or liposomes, orsalt-mediated aggregation of metallic (such as iron or gold)nanoparticles.

The multivalent molecules can have one or more labels (e.g., detectablereporter moieties). Examples of the labels include but are not limitedto fluorophores, spin labels, metals or metal ions, colorimetric labels,nanoparticles, PET labels, radioactive labels, or other such label asmay render said composition detectable by such methods as are known inthe art of the detection of macromolecules or molecular interactions.The label may be attached to the nucleotide (e.g. by attachment to thebase or the 5′ phosphate moiety of a nucleotide), to the particle itself(e.g., to the PEG subunits) or to the core (e.g., to the streptavidin oravidin core), to an end of the polymer, to a central moiety, or to anyother location within multivalent molecule which would be recognized byone of skill in the art to be sufficient to render said composition,such as a particle, detectable by such methods as are known in the artor described elsewhere herein. In some embodiments, a given multivalentmolecule is labeled with a known fluorophore to permit differentiationand identification of the nucleo-bases of the give multivalent molecule.

One example of the multivalent molecule is a polymer-nucleotideconjugate. Examples of the branched polymer include polyethylene glycol(PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid,polyglycolic acid, polyglycine, polyvinyl acetate, a dextran, or othersuch polymers. In one embodiment, the polymer is a PEG. In anotherembodiment, the polymer can have PEG branches.

Suitable polymers may be characterized by a repeating unit having afunctional group suitable for derivatization such as an amine, ahydroxyl, a carbonyl, or an allyl group. The polymer can also have oneor more pre-derivatized substituents such that one or more particularsubunits comprise a site of derivatization or a branch site, whether ornot other subunits include the same site, substituent, or moiety. Apre-derivatized substituent may comprise or may further comprise, forexample, a nucleotide, a nucleoside, a nucleotide analog, a label suchas a fluorescent label, radioactive label, or spin label, an interactionmoiety, an additional polymer moiety, or the like, or any combination ofthe foregoing.

In the multivalent molecule (e.g., polymer-nucleotide conjugate), thepolymer can have a plurality of branches. The branched polymer can havevarious configurations, including but are not limited to stellate(“starburst”) forms, aggregated stellate (“helter skelter”) forms,bottle brush, or dendrimer. The branched polymer can radiate from acentral attachment point or central moiety, or may include multiplebranch points, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morebranch points. In some embodiments, each subunit of a polymer mayoptionally constitute a separate branch point. An exemplary nucleotidearm is shown in FIG. 108 , and exemplary multivalent molecules are shownin FIGS. 104-107 .

In the multivalent molecule, the length and size of the branch candiffer based on the type of polymer. In some branched polymers, thebranch may have a length of between 1 and 1,000 nm, between 1 and 100nm, between 1 and 200 nm, between 1 and 300 nm, between 1 and 400 nm,between 1 and 500 nm, between 1 and 600 nm, between 1 and 700 nm,between 1 and 800 nm, or between 1 and 900 nm, or more, or having alength falling within or between any of the values disclosed herein. Insome branched polymers, the branch may have a size corresponding to anapparent molecular weight of 1K, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 30K,50K, 80K, 100K, or any value within a range defined by any two of theforegoing. The apparent molecular weight of a polymer may be calculatedfrom the known molecular weight of a representative number of subunits,as determined by size exclusion chromatography, as determined by massspectrometry, or as determined by any other method as is known in theart. The polymer can have multiple branches. The number of branches inthe polymer can be 2, 3, 4, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, ora number falling within a range defined by any two of these values.

For the multivalent molecule (e.g., polymer-nucleotide conjugate), thebranched polymer of 4, 8, 16, 32, or 64 branches can have nucleotidesattached to the ends of PEG branches, such that each end has attachedthereto 0, 1, 2, 3, 4, 5, 6 or more nucleotides. In one non-limitingexample, the branched polymer of between 3 and 128 PEG arms havingattached to the polymer branches ends one or more nucleotides, such thateach end has attached thereto 0, 1, 2, 3, 4, 5, 6 or more nucleotides ornucleotide analogs. In some embodiments, a branched polymer or dendrimerhas an even number of arms. In some embodiments, a branched polymer ordendrimer has an odd number of arms.

In the multivalent molecule (e.g., polymer-nucleotide conjugate), eachbranch or a subset of branches of the polymer may have attached theretoa moiety comprising a nucleotide (e.g., an adenine, a thymine, a uracil,a cytosine, or a guanine residue or a derivative or mimetic thereof),and the moiety is capable of binding to a polymerase, reversetranscriptase, or other nucleotide binding domain. Optionally, thenucleotide moiety may be capable of binding to apolymerase-template-primer complex but not incorporate, or canincorporate into an elongating nucleic acid chain during a polymerasereaction. In some embodiments, the nucleotide moiety comprises a chainterminating moiety which blocks incorporation of a subsequent nucleotideduring a polymerase-mediated reaction. In some embodiments, thenucleotide moiety may be unblocked (reversibly blocked) such that asubsequent nucleotide is not capable of being incorporated into anelongating nucleic acid chain during a polymerase reaction until suchblock is removed, after which the subsequent nucleotide is then capableof being incorporated into an elongating nucleic acid chain during apolymerase reaction.

The multivalent molecule can further have a binding moiety in eachbranch or a subset of branches. Some examples of the binding moietyinclude but are not limited to biotin, avidin, streptavidin or the like,polyhistidine domains, complementary paired nucleic acid domains,G-quartet forming nucleic acid domains, calmodulin, maltose-bindingprotein, cellulase, maltose, sucrose, glutathione-S-transferase,glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine andderivatives thereof, benzylcysteine and derivatives thereof, anantibody, an epitope, a protein A, a protein G. The binding moiety canbe any interactive molecules or fragment thereof known in the art tobind to or facilitate interactions between proteins, between proteinsand ligands, between proteins and nucleic acids, between nucleic acids,or between small molecule interaction domains or moieties.

In some embodiments, the multivalent molecule may comprise one or moreelements of a complementary interaction moiety. Exemplary complementaryinteraction moieties include, for example, biotin and avidin;SNAP-benzylguanosine; antibody or FAB and epitope; IgG FC and Protein A,Protein G, ProteinA/G, or Protein L; maltose binding protein andmaltose; lectin and cognate polysaccharide; ion chelation moieties,complementary nucleic acids, nucleic acids capable of forming triplex ortriple helical interactions; nucleic acids capable of formingG-quartets, and the like. One of skill in the art will readily recognizethat many pairs of moieties exist and are commonly used for theirproperty of interacting strongly and specifically with one another; andthus any such complementary pair or set is considered to be suitable forthis purpose in constructing or envisioning the compositions of thepresent disclosure. In some embodiments, a composition as disclosedherein may comprise compositions in which one element of a complementaryinteraction moiety is attached to one molecule or multivalent ligand,and the other element of the complementary interaction moiety isattached to a separate molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to a single molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to separate arms of, or locations on, a single molecule ormultivalent ligand. In some embodiments, a composition as disclosedherein may comprise compositions in which both or all elements of acomplementary interaction moiety are attached to the same arm of, orlocations on, a single molecule or multivalent ligand. In someembodiments, compositions comprising one element of a complementaryinteraction moiety and compositions comprising another element of acomplementary interaction moiety may be simultaneously or sequentiallymixed. In some embodiments, interactions between molecules or particlesas disclosed herein allow for the association or aggregation of multiplemolecules or particles such that, for example, detectable signals areincreased. In some embodiments, fluorescent, colorimetric, orradioactive signals are enhanced. In other embodiments, otherinteraction moieties as disclosed herein or as are known in the art arecontemplated. In some embodiments, a composition as provided herein maybe provided such that one or more molecules comprising a firstinteraction moiety such as, for example, one or more imidazole orpyridine moieties, and one or more additional molecules comprising asecond interaction moiety such as, for example, histidine residues, aresimultaneously or sequentially mixed. In some embodiments, saidcomposition comprises 1, 2, 3, 4, 5, 6, or more imidazole or pyridinemoieties. In some embodiments, said composition comprises 1, 2, 3, 4, 5,6, or more histidine residues. In such embodiments, interaction betweenthe molecules or particles as provided may be facilitated by thepresence of a divalent cation such as nickel, manganese, magnesium,calcium, strontium, or the like. In some embodiments, for example, a(His)3 group may interact with a (His)3 group on another molecule orparticle via coordination of a nickel or manganese ion.

The multivalent binding composition may comprise one or more buffers,salts, ions, or additives. In some embodiments, representative additivesmay include, but are not limited to, betaine, spermidine, detergentssuch as Triton X-100, Tween 20, SDS, or NP-40, ethylene glycol,polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol,methylcellulose, heparin, heparan sulfate, glycerol, sucrose,1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol,propylene glycol, polypropylene glycol, block copolymers such as thePluronic (r) series polymers, arginine, histidine, imidazole, or anycombination thereof, or any substance known in the art as a DNA“relaxer” (a compound, with the effect of altering the persistencelength of DNA, altering the number of within-polymer junctions orcrossings, or altering the conformational dynamics of a DNA moleculesuch that the accessibility of sites within the strand to DNA bindingmoieties is increased).

The multivalent molecules may include zwitterionic compounds asadditives. Further representative additives may be found in Lorenz, T.C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which is herebyincorporated by reference with respect to its disclosure of additivesfor the facilitation of nucleic acid binding or dynamics, or thefacilitation of processes involving the manipulation, use, or storage ofnucleic acids.

In some embodiments, the sequencing reaction comprises multivalentmolecules, polymerases, primed template molecules and at least onecation known in the art to facilitate nucleic acid interactions, such asself-association, secondary or tertiary structure formation, basepairing, surface association, peptide association, protein binding, orthe like. Examples of the cations include, but are not limited to,sodium, magnesium, strontium, barium, potassium, manganese, calcium,lithium, nickel, cobalt, or other such cations.

When the multivalent molecules are used to replace an unconjugated oruntethered nucleotide to form a complex with the polymerase and thenucleic acid template molecule, the local concentration of thenucleotide is increased many folds, which in turn enhances the signalintensity, particularly the correct signal versus mismatch. The presentdisclosure contemplates contacting the multivalent binding compositionwith a polymerase and a primed template molecule to determine theformation of a ternary binding complex.

Because of the increased local concentration of the nucleotide on thepolymer-nucleotide conjugate, the binding between the polymerase, theprimed template strand, and the nucleotide, when the nucleotide iscomplementary to the next base of the template strand, becomes morefavorable. The formed binding complex has a longer persistence timewhich in turn helps shorten the imaging step. The high signal intensityresulted from the use of the polymer nucleotide conjugate remain for theentire binding and imaging step. The strong binding between thepolymerase, the primed template strand, and the nucleotide or nucleotideanalog also means that the formed binding complex will remain stabilizedduring the washing step and the signal will remain at a high intensitywhen other reaction mixture and unmatched nucleotide analogs are washedaway. After the imaging step, the binding complex can be destabilizedand the primed template strand can then be extended for one base. Afterthe extension, the binding and imaging steps can be repeated again withthe use of the polymer nucleotide conjugate to determine the identity ofthe next base.

The compositions and methods of the present disclosure provide a robustand controllable means of establishing and maintaining a ternary enzymecomplex (e.g., during sequencing), as well as providing vastly improvedmeans by which the presence of said complex may be identified and/ormeasured, and a means by which the persistence of said complex may becontrolled. This provides important solutions to problems such as thatof determining the identity of the N+1 base in nucleic acid sequencingapplications.

Without intending to be bound by any particular theory, it has beenobserved that multivalent binding compositions disclosed hereinassociate with polymerase nucleotide complexes in order to form aternary binding complexes with a rate that is time-dependent, thoughsubstantially slower than the rate of association known to be obtainableby nucleotides in free solution. Thus, the on-rate (Kon) issubstantially and surprisingly slower than the on rate for singlenucleotides or nucleotides not attached to multivalent ligand complexes.Importantly, however, the off rate (Koff) of the multivalent ligandcomplex is substantially slower than that observed for nucleotides infree solution. Therefore, the multivalent ligand complexes of thepresent disclosure provide a surprising and beneficial improvement ofthe persistence of ternary polymerase-polynucleotide-nucleotidecomplexes (especially over such complexes that are formed with freenucleotides) allowing, for example, significant improvements in imagingquality for nucleic acid sequencing applications, over currentlyavailable methods and reagents. Importantly, this property of themultivalent substrates disclosed herein renders the formation of visibleternary complexes controllable, such that subsequent visualization,modification, or processing steps may be undertaken essentially withoutregard to the dissociation of the complex—that is, the complex can beformed, imaged, modified, or used in other ways as necessary, and willremain stable until a user carries out an affirmative dissociation step,such as exposing the complexes to a dissociation buffer.

Compositions and methods for preparing and using the multivalentmolecules (also called polymer-nucleotide conjugates) are described inU.S. Ser. No. 16/579,794, filed on Sep. 23, 2019, the contents of whichis hereby expressly incorporated by reference in its entirety.

Sequencing Polymerases

The present disclosure provides compositions and methods for pairwisesequencing, including methods for sequencing the plurality ofimmobilized concatemer template molecules and methods for sequencing theplurality of retained forward extension strands, where any of thesequencing methods described herein employ at least one type ofsequencing polymerase and a plurality of nucleotides. In someembodiments, the sequencing polymerase(s) is/are capable ofincorporating a complementary nucleotide opposite a nucleotide having ascissile moiety in the immobilized concatemer template molecules. Insome embodiments, the plurality of sequencing polymerases compriserecombinant sequencing polymerases.

Examples of suitable polymerases for use in sequencing with nucleotidesinclude but are not limited to: Klenow DNA polymerase; Thermus aquaticusDNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatusaltiarchaeales archaeon; Candidatus Hadarchaeum Yellowstonense;Hadesarchaea archaeon; Euryarchaeota archaeon; Thermoplasmata archaeon;Thermococcus polymerases such as Thermococcus litoralis, bacteriophageT7 DNA polymerase; human alpha, delta and epsilon DNA polymerases;bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNApolymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase);Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alphaand epsilon; 9 degree N polymerase; reverse transcriptases such as HIVtype M or O reverse transcriptases; avian myeloblastosis virus reversetranscriptase; Moloney Murine Leukemia Virus (MMLV) reversetranscriptase; or telomerase. Further non-limiting examples of DNApolymerases include those from various Archaea genera, such as,Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus,Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus,Thermococcus, and Vulcanisaeta and the like or variants thereof,including such polymerases as are known in the art such as 9 degrees N,VENT, DEEP VENT, THERMINATOR, Pfu, KOD, Pfx, Tgo and RB69 polymerases.

The present disclosure provides compositions and methods for pairwisesequencing, including methods for sequencing the plurality ofimmobilized concatemer template molecules and methods for sequencing theplurality of retained forward extension strands, where any of thesequencing methods described herein employ a first type of sequencingpolymerase and a plurality of multivalent molecules, and a second typeof sequencing polymerase and a plurality of nucleotides. In someembodiments, the first sequencing polymerases are capable of binding acomplementary nucleotide unit of a multivalent molecule opposite anucleotide having a scissile moiety (e.g., uridine, 8-oxoG ordeoxyinosine) in the immobilized concatemer template molecules. In someembodiments, the second sequencing polymerases are capable ofincorporating a complementary nucleotide opposite a nucleotide having ascissile moiety (e.g., uridine, 8-oxoG or deoxyinosine) in theimmobilized concatemer template molecules. In some embodiments, theplurality of first and second sequencing polymerases compriserecombinant sequencing polymerases. In some embodiments, the pluralityof first and second sequencing polymerases comprise one, two or moresubstitution mutations that confer an exonuclease minus property.

Examples of suitable first and/or second sequencing polymerases for usein sequencing with multivalent molecules and nucleotides include but arenot limited to: Klenow DNA polymerase; Thermus aquaticus DNA polymeraseI (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaealesarchaeon; Candidatus Hadarchaeum Yellowstonense; Hadesarchaea archaeon;Euryarchaeota archaeon; Thermoplasmata archaeon; Thermococcuspolymerases such as Thermococcus litoralis, bacteriophage T7 DNApolymerase; human alpha, delta and epsilon DNA polymerases;bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNApolymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase);Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alphaand epsilon; 9 degree N polymerase; reverse transcriptases such as HIVtype M or O reverse transcriptases; avian myeloblastosis virus reversetranscriptase; Moloney Murine Leukemia Virus (MMLV) reversetranscriptase; or telomerase. Further non-limiting examples of DNApolymerases include those from various Archaea genera, such as,Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus,Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus,Thermococcus, and Vulcanisaeta and the like or variants thereof,including such polymerases as are known in the art such as 9 degrees N,VENT, DEEP VENT, THERMINATOR, Pfu, KOD, Pfx, Tgo and RB69 polymerases.

In some embodiments, the plurality of first and/or second sequencingpolymerases comprise mutant polymerases that exhibit increasedincorporation rate of nucleotide analogs and/or increased fidelitycompared to a wild type polymerase. In some embodiments, the nucleotideanalogs comprise a chain terminating moiety (e.g., blocking moiety) atthe sugar 2′ position and/or at the 3′ sugar position. In someembodiments, the plurality of first and/or second sequencing polymerasesexhibit increased thermal stability compared to a wild type polymerase.In some embodiments, the plurality of first and/or second sequencingpolymerases exhibit increased ability to incorporate a nucleotide intothe 3′ end of a primer or incorporate a nucleotide into a nascent strandin a primer extension reaction when the template molecule includes atleast one uridine (e.g., uracil-tolerant sequencing polymerase). In someembodiments, the first polymerases have the same or different amino acidsequence as the second polymerases.

In some embodiments, polymerases that are suitable for conducting asequencing reaction include polymerases that can bind a nucleic acidduplex (e.g., a template molecule hybridized to a primer) to form acomplexed polymerase, where the complexed polymerase can bind anucleotide to form a binding complex. The binding complex can transitionto become a ternary complex.

In some embodiments, polymerases (e.g., first polymerases) that aresuitable for conducting a sequencing reaction include polymerases thatcan bind a nucleic acid duplex (e.g., a template molecule hybridized toa primer) to form a complexed polymerase, where the complexed polymerasecan bind a nucleotide unit of a multivalent molecule to form a bindingcomplex. The binding complex can transition to become a ternary complex.

In some embodiments, polymerases (e.g., second polymerases) that aresuitable for conducting a sequencing reaction include polymerases thatcan bind a nucleic acid duplex (e.g., a template molecule hybridized toa primer) to form a complexed polymerase, where the complexed polymerasecan bind a nucleotide to form a binding complex. The binding complex cantransition to become a ternary complex.

Any suitable sequencing polymerase described herein can form a bindingcomplex that can transition into a ternary complex when the freenucleotide, or nucleotide unit of a multivalent molecule, binds to the3′ end of the nucleic acid primer (as part of the nucleic acid duplex)at a position that is opposite a complementary nucleotide in the nucleicacid template molecule.

The ternary complex has a longer persistence time when the nucleotideunit of a multivalent molecule is complementary to the target nucleicacid compared to the persistence time of a non-complementary nucleotideunit. The ternary complex also has a longer persistence time when anucleotide unit of a multivalent molecule is complementary to the targetnucleic acid compared to a complementary free nucleotide. In someembodiments, the ternary complexes have a persistence time of greaterthan about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 second or morethan 30 seconds. For example, in some embodiments, the ternary complexeshave a persistence time of greater than about 0.1-0.25 seconds, or about0.25-0.5 seconds, or about 0.5-0.75 seconds, or about 0.75-1 second, orabout 1-2 seconds, or about 2-3 seconds, or about 3-4 second, or about4-5 seconds, or about 5-30 seconds or more than 30 seconds and/orwherein the method is or may be carried out at a temperature of at orabove 15° C., at or above 20° C., at or above 25° C., at or above 35°C., at or above 37° C., at or above 42° C. at or above 55° C. at orabove 60° C., or at or above 72° C., or within a range defined by any ofthe foregoing. In some embodiments, the ternary complexes may have apersistence time of less than 1 s, greater than 1 s, greater than 2 s,greater than 3 s, greater than 5 s, greater than 10 s, greater than 15s, greater than 20 s, greater than 30 s, greater than 60 s, greater than120 s, greater than 360 s, greater than 3600 s, or more, or for a timelying within a range defined by any two or more of these values. Theternary complex (e.g., binding complex) remains stable until subjectedto a condition that causes dissociation of interactions between any ofthe polymerase, template molecule, primer and/or the nucleotide unit orthe nucleotide. For example, a dissociating condition comprisescontacting the binding complex with any one or any combination of adetergent, EDTA and/or water.

The persistence time can be measured, for example, by observing theonset and/or duration of a ternary complex, such as by observing asignal from a labeled component of the ternary complex. For example, alabeled nucleotide or a labeled reagent comprising one or morenucleotides may be present in a ternary complex, thus allowing thesignal from the label to be detected during the persistence time of theternary complex.

It has been observed that different ranges of persistence times areachievable with different salts or ions, showing, for example, thatcomplexes formed in the presence of, for example, magnesium or manganeseform more quickly than complexes formed with other ions. It has alsobeen observed that complexes formed in the presence of, for example,strontium or barium, form readily and dissociate completely or withsubstantial completeness upon withdrawal of strontium or barium, or uponwashing with a buffer lacking strontium or barium.

The dissociation of ternary complexes can be controlled by changing thebuffer conditions. During a sequencing method, an imaging step can beused to detect and/or identify a nucleotide unit bound to a nucleic acidduplex in a ternary complex. After the imaging step, a buffer withincreased salt content can be used to dissociate the ternary complexessuch that labeled multivalent molecules can be washed out, providing ameans by which signals can be attenuated or terminated, such as in thetransition between one sequencing cycle and the next. This dissociationmay be effected, in some embodiments, by washing the complexes with abuffer lacking a necessary metal or cofactor. In some embodiments, awash buffer may comprise one or more reagents for the purpose ofmaintaining pH control. In some embodiments, a wash buffer may compriseone or more monovalent cations, such as sodium. In some embodiments, awash buffer lacks or substantially lacks a divalent cation, for example,having no or substantially no strontium, barium, calcium, magnesium, ormanganese. In some embodiments, a wash buffer further comprises achelating agent, such as, for example, EDTA, EGTA, nitrilotriaceticacid, polyhistidine, imidazole, or the like. In some embodiments, a washbuffer may maintain the pH of the environment at the same level as forthe ternary complex. In some embodiments, a wash buffer may raise orlower the pH of the environment relative to the level seen for theternary complex. In some embodiments, the pH may be within a range from2-4, 2-7, 5-8, 7-9, 7-10, or lower than 2, or higher than 10, or a rangedefined by any two of the values provided herein.

Addition of a particular ion may affect the binding of the polymerase toa primed target nucleic acid, the formation of a ternary complex, thedissociation of a ternary complex, or the incorporation of one or morenucleotides into an elongating nucleic acid such as during a polymerasereaction. In some embodiments, relevant anions may comprise chloride,acetate, gluconate, sulfate, phosphate, or the like. In someembodiments, an ion may be included in a binding buffer including one ormore acids, bases, or salts, such as NiCl₂, CoCl₂, MgCl₂, MnCl₂, SrCl₂,CaCl₂, CaSO₄, SrCO₃, BaCl₂ or the like. Representative salts, ions,solutions and conditions may be found in Remington: The Science andPractice of Pharmacy, 20th. Edition, Gennaro, A. R., Ed. (2000), whichis hereby incorporated by reference in its entirety, and especially withrespect to Chapter 17 and related disclosure of salts, ions, saltsolutions, and ionic solutions.

The binding between a nucleic acid template molecule and a multivalentmolecule can be conducted in the presence of a sequencing polymerasethat has been rendered catalytically inactive. In some embodiments, thesequencing polymerase has been rendered catalytically inactive bymutation. In some embodiments, the sequencing polymerase has beenrendered catalytically inactive by chemical modification. In someembodiments, the sequencing polymerase has been rendered catalyticallyinactive by the absence of a necessary substrate, ion, or cofactor. Insome embodiments, the sequencing polymerase has been renderedcatalytically inactive by the absence of magnesium or manganese ions.

The binding between a nucleic acid template molecule and a multivalentmolecule can occur in the presence of a sequencing polymerase whereinthe binding solution lacks a catalytic ion such as magnesium ormanganese. A catalytic ion can promote polymerase-catalyzedincorporation of a nucleotide or nucleotide unit. Alternatively, thebinding between a nucleic acid template molecule and a multivalentmolecule can occur in the presence of a sequencing polymerase whereinthe binding solution comprises a non-catalytic ion such strontium,barium or calcium. A non-catalytic ion can inhibit polymerase-catalyzedincorporation of a nucleotide or nucleotide unit.

A non-catalytic divalent cation (e.g., strontium or barium) can promoteformation of a stable ternary complex having a polymerase bound to aprimed nucleic acid template molecule and a multivalent molecule (e.g.,fluorescently labeled multivalent molecule), and inhibitpolymerase-catalyzed incorporation of the nucleotide unit, where thestable ternary complex has a persistence time that permits detection andimaging by fluorescence detection or by other methods known in the art.Unbound polymer-nucleotide conjugates may optionally be washed awayprior to detection of the ternary binding complex.

A catalytic divalent cation (e.g., magnesium or manganese) can promoteformation of a stable ternary complex having a polymerase bound to aprimed nucleic acid template molecule and a free nucleotide (e.g.,fluorescently labeled nucleotide), and promote polymerase-catalyzedincorporation of the nucleotide, where the stable ternary complex has apersistence time that permits detection and imaging by fluorescencedetection or by other methods known in the art.

Cleaving Reagents

The present disclosure provides compositions and methods for pairwisesequencing, including methods for cleaving/removing a chain terminatormoiety from an incorporated terminal nucleotide on a nascent strandduring a sequencing workflow. For example, the incorporated terminalnucleotide comprises a chain terminator moiety linked to the 3′ sugarposition by a cleavable linker. In some embodiments, the cleavablelinker can be cleaved using a cleaving reagent.

The present disclosure also provides compositions and methods forpairwise sequencing, including methods for cleaving/removing adetectable reporter moiety (e.g., a fluorophore) from an incorporatednucleotide on a nascent strand during a sequencing workflow. Forexample, the incorporated nucleotide comprises a fluorophore linked tothe nucleo-base. In some embodiments, the linker that attaches thefluorophore to the nucleo-base is cleavable under the same conditionthat will cleave the cleavable linker at the 3′ sugar position.

The present disclosure also provides compositions and methods forpairwise sequencing, including methods for cleaving/removing a chainterminator moiety from a multivalent molecule that is bound and/orunbound to a complexed polymerase using a cleaving reagent under acondition suitable for cleaving the chain terminator moiety from thenucleotide units of the multivalent molecule.

The present disclosure also provides compositions and methods forpairwise sequencing, including methods for cleaving/removing adetectable reporter moiety (e.g., fluorophore) from a multivalentmolecule that is bound and/or unbound to a complexed polymerase using acleaving reagent under a condition suitable for cleaving the detectablereporter moiety from the nucleotide units of the multivalent molecule.

In some embodiments, the cleaving reagent comprises at least onecleaving compound that cleaves the linker that attaches the fluorophoreto the nucleotide or to the nucleotide unit of a multivalent molecule.

In some embodiments, the cleaving reagent comprises at least onecleaving compound that cleaves the linker that attaches the chainterminator moiety to the nucleotide or to the nucleotide unit.

In some embodiments, the cleaving compound comprises piperidine,2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), ortetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄), with piperidine.

In some embodiment, the cleaving compound comprises a palladiumcatalyst, for example palladium-on-carbon (Pd/C).

In some embodiments, the cleaving compound comprisesbeta-mercaptoethanol or dithiothritol (DTT).

In some embodiments, the cleaving compound comprise potassium carbonate(K₂CO₃) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH).

In some embodiments, the cleaving compound comprises tetrabutylammoniumfluoride, pyridine-HF, ammonium fluoride, or triethylaminetrihydrofluoride.

In some embodiments, the cleaving compound comprises a phosphinecompound, including for example a phosphine having a derivatizedtri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety.In some embodiments, the phosphine cleaving compound comprisesTris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine(BS-TPP). In some embodiments, the phosphine cleaving compound comprisesTri(hydroxyproyl)phosphine (THPP) or Tris(hydroxymethyl)phosphine (THMP)or 4-dimethylaminopyridine (4-DMAP).

In some embodiments, the cleaving catalyst comprises4-dimethylaminopyridine (4-DMAP).

The cleaving reagents can include a cleaving compound and/or a cleavingcatalyst at a concentration of about 1-10 mM, or about 10-25 mM, orabout 25-50 mM, or about 50-75 mM, or about 75-100 mM.

In some embodiments, the cleaving reagent can include a pH bufferingagent. The pH buffering agent comprises any one or any combination oftwo or more of Tris, Tris-HCl, Tricine, Bicine, Bis-Tris propane, HEPES,MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine(a.k.a 2-amino methanol; MEA), a citrate compound, a citrate mixture,NaOH and/or KOH. In some embodiments, the pH buffering agent can bepresent in cleaving reagent at a concentration of about 1-100 mM, orabout 10-50 mM, or about 10-25 mM. In some embodiments, the pH of the pHbuffering agent which is present in the cleaving reagent can be adjustedto a pH of about 4-9.5, or a pH of about 5-9.5, or a pH of about 6-8.5.

In some embodiments, the cleaving reagent can include a monovalentcation which comprises sodium or potassium. In some embodiments, themonovalent cation is in the form of MgCl₂ or NaCl. In some embodiments,the cleaving reagent comprises MgCl₂ at a concentration of about 1-10mM, or about 10-20 mM, or about 10-50 mM. In some embodiments, thecleaving reagent comprises NaCl at a concentration of about 25-200 mM,or about 50-150 mM.

in some embodiments, cleaving reagent can include a detergent. In someembodiments, the detergent comprises an ionic detergent such as SDS(sodium dodecyl sulfate). In some embodiments, the detergent comprises anon-ionic detergent such as Triton X-100, Tween 20, Tween 80 or NonidetP-40. In some embodiments, the detergent comprises a zwitterionicdetergent such as CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) orN-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX). In someembodiments, the detergent comprises LDS (dodecyl sulfate), sodiumtaurodeoxycholate, sodium taurocholate, sodium glycocholate, sodiumdeoxycholate or sodium cholate. In some embodiments, the detergent isincluded in a cleaving reagent at a concentration of about 0.01-0.05%,or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%, or about0.2-0.25%.

Cleaving Reagents with Compounds to Reduce Photo-Damage

The various pairwise sequencing workflows described herein generallyinclude conducting a forward sequencing reaction to generate a pluralityof extended forward sequencing primer strands, and conducting a reversesequencing reaction to generate a plurality of extended reversesequencing primer strands. The forward and reverse sequencing reactionscan be conducted using fluorescently-labeled nucleotides, or usingfluorescently-labeled multivalent molecules and nucleotides (labeledand/or unlabeled nucleotide). Detection and identification of thefluorescently-labeled nucleotides and the fluorescently-labeledmultivalent molecules during a forward and reverse sequencing reactionsyields forward reads (e.g., R1 reads) and reverse reads (e.g., R2reads), respectively. The detection step includes exciting thefluorophores with radiation energy (e.g., light) and emission of afluorescent signal from the fluorophores. Typically, high intensityfluorescent signals during the forward and reverse reads increasesequencing accuracy, while low intensity signal negatively impact theaccuracy of the forward and reverse reads. Ideally, the intensity of theforward and reverse reads are similar to each other, and are notaffected by the read length (e.g., number of sequencing cycles) of theforward sequencing reads. In a non-ideal situation, the intensity of thereverse sequencing reads (R2 reads) drops when the number of forwardsequencing reads increases. Without wishing to be bound to theory, it ispostulated that a decrease in signal intensity of the reverse sequencingreads when the forward sequencing reads are long (e.g., longer than 5-10sequencing cycles) is caused by photo damage to the immobilizedconcatemer template molecules and/or the retained forward extensionstrand. When the cleaving reagent includes at least one compound thatreduces photo-damage to the nucleic acids, the signal intensity of thereverse sequencing reads increases even for long forward sequencingreads (e.g., see FIG. 119 ).

The present disclosure provides cleaving reagents comprising: at leastone solvent; a pH buffering agent; at least one monovalent cation; adetergent; a cleaving compound and/or a cleaving catalyst; and at leastone or any combination of two or more compounds that can reducephoto-damage, including antioxidants, triplet state quenchers, singletoxygen quenchers, oxygen scavengers, electron scavengers, anti-fadeformulations. The skilled artisan will appreciate that some of thesecompounds can be classified as more than one type of photo-damagereducing compound.

In some embodiments, the cleaving reagent can include at least oneantioxidant, including ascorbic acid, ascorbyl palmitate, D-isoascorbicacid (erythorbic acid), sodium ascorbate, butylated hydroxytoluene(BTH), butylated hydroxy toluene (BHT), polyphenol antioxidants,polyvinyl alcohols, butylated hydroxy anisol (BHA) or Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or othervitamin E analogs including nitrated Trolox derivates (see U.S. Pat. No.9,994,541, the entire contents of which are expressly incorporated byreference in its entirety).

In some embodiments, the cleaving reagent can include at least onetriplet state quenchers including ascorbic acid,1,4-diazobicyclo[2.2.2]octane (DABCO), cyclo-octatetraene (COT),dithiothreitol (DTT), mercaptoethylamine (MEA), β-mercaptoethanol (BME),n-propyl gallate, p-phenylenediamene (PPD), hydroquinone and sodiumazide (NaN₃), TEMPO (2,2,6,6-tetramethyl1-1-piperidinyloxyl), HTEMPO(4-hydroxy derivative of TEMPO) and/or DTBN (di-t-butylnitroxide).

In some embodiments, the cleaving compound can include at least onesinglet oxygen quenchers including thiol-based quenchers such asglutathione, dithiothreitol, ergothioneine, methionine, cysteine,beta-dimethyl cysteine (penicillamine), mercaptopropionylglycine, MESNA,imidazole, and/or N-acetyl cysteine and captopril.

In some embodiments, the cleaving compound can include at least oneoxygen scavenger including glutathione, and N-acetylcysteine, histidine,tryptophan, hydrazine (N₂H₄), sodium sulfite (Na₂SO₃) and/orhydroxylamine.

In some embodiments, the cleaving compound can include at least oneelectron scavenger including methyl viologen (e.g.,1,1′-dimethyl-4,4′-bipyridinium dichloride).

In some embodiments, the cleaving compound can include at least oneanti-fade formulation including commercially-available productsincluding Fluoroguard Antifade Reagent (e.g., from BioRad), SlowFadeAntifade Kit (e.g., includes DABCO, from Molecular Probes-Invitrogen),ProLong Gold Antifade Reagent (e.g., from Invitrogen), and/or CitiFluor(e.g., from CitiFluor).

The cleaving reagent can include at least one of the compounds thatreduce photo damage at a concentration of about 0.1-1 mM, or about 1-10mM, or about 10-25 mM, or about mM, or about 50-75 mM, or about 75-100mM.

High Efficiency Hybridization Buffers

The present disclosure provides pairwise sequencing compositions andmethods which employ a high efficiency hybridization buffer. The highefficiency hybridization buffer can be used to hybridize soluble forwardsequencing primers (e.g., second plurality of forward sequencingprimers) to the retained immobilized concatemer template molecules. Thehigh efficiency hybridization buffer can be used to hybridize solublereverse sequencing primers to the retained forward extension strands.

The high efficiency hybridization buffers described herein promote highstringency (e.g., specificity), speed, and efficacy of nucleic acidhybridization reactions and increases the efficiency of the subsequentamplification and sequencing steps. The high efficiency hybridizationbuffers can significantly shorten nucleic acid hybridization times, anddecreases sample input requirements. The high efficiency hybridizationbuffers can be used for nucleic acid annealing workflows at isothermalconditions which eliminates requirement of a cooling step for annealing.

In some embodiments, the high efficiency hybridization buffer comprisesa first and second polar aprotic solvent, a pH buffer system and acrowding agent. The polar solvent can be a solvent or solvent systemcomprising one or more molecules characterized by the presence of apermanent dipole moment, i.e., a molecule having a spatially unequaldistribution of charge density. A polar solvent may be characterized bya dielectric constant of 20, 25, 30, 35, 40, 45, 50, 55, 60 or by avalue or a range of values having any of the aforementioned values. Apolar solvent may comprise a polar aprotic solvent. A polar aproticsolvent may further contain no ionizable hydrogen in the molecule. Inaddition, polar solvents or polar aprotic solvents may be preferablysubstituted in the context of the presently disclosed compositions witha strong polarizing functional groups such as nitrile, carbonyl, thiol,lactone, sulfone, sulfite, and carbonate groups so that the underlyingsolvent molecules have a dipole moment. Polar solvents and polar aproticsolvents can be present in both aliphatic and aromatic or cyclic form.In some embodiments, the polar solvent is acetonitrile.

In some embodiments, the polar or polar aprotic solvent can have adielectric constant that is the same as or close to acetonitrile. Thedielectric constant of the polar or polar aprotic solvent can be in therange of about 20-60, about 25-55, about 25-50, about 25-45, about25-40, about 30-50, about 30-45, or about 30-40. The dielectric constantof the polar or polar aprotic solvent can be greater than 20, 25, 30,35, or 40. The dielectric constant of the polar or polar aprotic solventcan be lower than 30, 40, 45, 50, 55, or 60. The dielectric constant ofthe polar or polar aprotic solvent can be about 35, 36, 37, 38, or 39.

In some embodiments, the polar or polar aprotic solvent described hereincan have a polarity index that is the same as or close to acetonitrile.The polarity index of the polar or polar aprotic solvent can be in therange of about 2-9, 2-8, 2-7, 2-6, 3-9, 3-8, 3-7, 3-6, 4-9, 4-8, 4-7, or4-6. The polarity index of the polar or polar aprotic solvent can begreater than about 2, 3, 4, 4.5, 5, 5.5, or 6. The polarity index of thepolar or polar aprotic solvent can be lower than about 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, or 10. The polarity index of the polar or polaraprotic solvent can be about 5.5, 5.6, 5.7, or 5.8.

Some examples of the polar or polar aprotic solvent include but are notlimited to acetonitrile, dimethylformamide (DMF), dimethylsulfoxide(DMSO), acetanilide, N-acetyl pyrrolidone, 4-amino pyridine, benzamide,benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylenecarbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleicanhydride, 2-chlorocyclohexanone, chloroethylene carbonate,chloronitromethane, citraconic anhydride, crotonlactone,5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethylsulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone,epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate,N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate,ethylene glycol sulfate, ethylene glycol sulfite, furfural,2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxybenzonitrile, 1-methoxy-2-nitrobenzene, methyl alpha bromo tetronate,1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methylmorpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone,methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline,nitrobenzimidazole, 2-nitrofuran, 1-nitroso-2-pyrolidinone,2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine),1,3-propane sultone, β-propiolactone, propylene carbonate,4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone,saccharin, succinonitrile, sulfanilamide, sulfolane,2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide,tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil,3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3-trichloropropene, trimethylene sulfide-dioxide, and trimethylene sulfite.

In some embodiments, the high efficiency hybridization buffer includesan amount of the polar solvent or polar aprotic solvent in an amounteffective to denature a double stranded nucleic acid. In someembodiments, the amount of the polar or polar aprotic solvent is greaterthan about 10% by volume based on the total volume of the formulation.The amount of the polar or polar aprotic solvent is about or more thanabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher, by volume based on the total volume of the formulation. Theamount of the polar or polar aprotic solvent is lower than about 15%,20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher, by volumebased on the total volume of the formulation. In some embodiments, theamount of the polar or polar aprotic solvent is in the range of about10% to 90% by volume based on the total volume of the formulation. Insome embodiments, the amount of the polar or polar aprotic solvent is inthe range of about 25% to 75% by volume based on the total volume of theformulation. In some embodiments, the amount of the polar or polaraprotic solvent is in the range of about 10% to 95%, 10% to 85%, 20% to90%, 20% to 80%, 20% to 75%, or 30% to 60% by volume based on the totalvolume of the formulation.

In some embodiments, the high efficiency hybridization buffer mayinclude an organic solvent. Examples of suitable solvents include, butare not limited to, acetonitrile, ethanol, DMF, and methanol, or anycombination thereof at varying percentages (typically >5%). In someembodiments, the percentage of organic solvent (by volume) included inthe high efficiency hybridization buffer may range from about 1% toabout 20%. In some embodiments, the percentage by volume of organicsolvent may be at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 15%, or at least 20%. In some embodiments, the percentageby volume of organic solvent may be at most 20%, at most 15%, at most10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most4%, at most 3%, at most 2%, or at most 1%. Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of organic solvent may range from about 4% to about 15%. Those ofskill in the art will recognize that the percentage by volume of organicsolvent may have any value within this range, e.g., about 7.5%.

In some embodiments, the use of a high efficiency hybridization buffercan improve nucleic acid hybridization rates. When a high efficiencyhybridization buffer is used in combination with a support having a lownon-specific binding coating, the relative hybridization rates mayimprove and can range from about 2× to about 20× faster than that for aconventional hybridization protocol using a conventional hybridizationbuffer with a support having a conventional coating. In someembodiments, the use of a high efficiency hybridization buffer canimprove the relative hybridization rates of nucleic acid by at least 2×,at least 3×, at least 4×, at least 5×, at least 6×, at least 7×, atleast 8×, at least 9×, at least 10×, at least 12×, at least 14×, atleast 16×, at least 18×, or at least 20×, compared to that for aconventional hybridization buffer protocol.

Improvements in hybridization efficiency (or yield) is a measure of thepercentage of total available tethered adaptor sequences (e.g., surfaceprimers) on a solid surface, primer sequences, or oligonucleotidesequences in general that are hybridized to complementary sequences. Insome embodiments, the use of a high efficiency hybridization buffer(optionally, used in combination with low non-specific binding surface)yield improved hybridization efficiency compared to that for aconventional hybridization protocol. In some embodiments, thehybridization efficiency that may be achieved is better than 80%, 85%,90%, 95%, 98%, or 99% in any of the hybridization reaction timesspecified above.

Improvements in hybridization specificity is a measure of the ability oftethered adapter sequences (e.g., surface primers), primer sequences, oroligonucleotide sequences in general to correctly hybridize selectivelyto complementary sequences. In some embodiments, the use of a highefficiency hybridization buffer (optionally, used in combination withlow non-specific binding surface) yield improved hybridizationspecificity compared to that for a conventional hybridization protocol.In some embodiments, the hybridization specificity that may be achievedis better than 1 base mismatch in 10 hybridization events, 1 basemismatch in 100 hybridization events, 1 base mismatch in 1,000hybridization events, or 1 base mismatch in hybridization events.

The term “crowding agent” and related terms refers to a compound thatalters the properties of other molecules in a solution. Crowding agentstypically have high molecular weight and/or bulky structures. Crowdingagents in solution can increase the concentration of other molecules inthe solution. Crowding agents can reduce the volume of solvent that isavailable for other molecules in the solution which can create amolecular crowding environment. Crowding agents in a solution cangenerate a crowded environment for molecules in the solution. Crowdingagents can alter the rates or equilibrium constants of a reaction.Examples of crowding agents include polyethylene glycol (e.g., PEG),ficoll, dextran, glycogen, polyvinyl alcohol, triblock polymers (e.g.,Pluronics), polystyrene, polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC),hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methycellulose,and hydroxyl methyl cellulose. In some embodiments, the crowding agentcomprises linear or branched polyethylene glycol (PEG). In someembodiments, the crowding agent comprise PEG 400, PEG 1500, PEG 2000,PEG 3400, PEG 3350, PEG 4000, PEG 6000 or PEG 8000. In some embodiments,a high efficiency hybridization buffer can include at least one crowdingagent at about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,60%, or higher percent based on volume of the solution. In someembodiments, high efficiency hybridization buffer can be used for anucleic acid sequencing reaction, or for nucleic acid amplificationincluding rolling circle amplification and/or multiple displacementamplification reactions.

The high efficiency hybridization buffer includes an amount of acrowding agent that will permit, enhances, or facilitates molecularcrowding. The amount of the crowding agent is about 1%, 2%, 3%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or higher amounts, by volumebased on the total volume of the high efficiency hybridization bufferformulation. In some embodiments, the amount of a molecular crowdingagent is greater than 5% by volume based on the total volume of the highefficiency hybridization buffer formulation. The amount of the crowdingagent is lower than about 3%, 5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%,40%, 50%, 60%, 70%, 80%, 90%, or more than 90%, by volume based on thetotal volume of the high efficiency hybridization buffer formulation. Insome embodiments, the amount of the molecular crowding agent can be lessthan 30% by volume based on the total volume of the formulation. In someembodiments, the amount of the polar or polar aprotic solvent is in therange of about 25% to 75% by volume based on the total volume of theformulation. In some embodiments, the amount of the polar or polaraprotic solvent is in the range of about 1% to 40%, 1% to 35%, 2% to50%, 2% to 40%, 2% to 35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%,5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, byvolume based on the total volume of the formulation. In some cases, theamount of the molecular crowding agent can be in the range of about 5%to about 20% by volume based on the total volume of the formulation. Insome embodiments, the amount of the crowding agent is in the range ofabout 1% to 30% by volume based on the total volume of the formulation.

In some embodiments, the high efficiency hybridization bufferformulations may include the addition of a molecular crowding or volumeexclusion agent. Molecular crowding or volume exclusion agents aretypically macromolecules (e.g., proteins) which, when added to asolution in high concentrations, may alter the properties of othermolecules in solution by reducing the volume of solvent available to theother molecules. In some embodiments, the percentage by volume ofmolecular crowding or volume exclusion agent included in the highefficiency hybridization buffer formulation may range from about 1% toabout 50%. In some embodiments, the percentage by volume of molecularcrowding or volume exclusion agent may be at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50%. In someembodiments, the percentage by volume of molecular crowding or volumeexclusion agent may be at most 50%, at most 45%, at most 40%, at most35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, atmost 5%, or at most 1%. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, the percentage by volume of molecularcrowding or volume exclusion agent may range from about 5% to about 35%.Those of skill in the art will recognize that the percentage by volumeof molecular crowding or volume exclusion agent may have any valuewithin this range, e.g., about 12.5%.

In some embodiments, the high efficiency hybridization buffer includes apH buffer system that maintains the pH of the compositions in a rangesuitable for hybridization process. The pH buffer system can include oneor more buffering agents selected from the group consisting of Tris,HEPES, TAPS, Tricine, Bicine, Bis-Tris, NaOH, KOH, TES, EPPS, MES, andMOPS. The pH buffer system can further include a solvent. A preferred pHbuffer system includes MOPS, MES, TAPS, phosphate buffer combined withmethanol, acetonitrile, ethanol, isopropanol, butanol, t-butyl alcohol,DMF, DMSO, or any combination therein.

In some embodiments, the high efficiency hybridization buffer includesan amount of the pH buffer system that is effective to maintain the pHof the formulation to be in a range suitable for the hybridization. Insome embodiments, the pH may be at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, or at least 10. In someembodiments, the pH may be at most 10, at most 9, at most 8, at most 7,at most 6, at most 5, at most 4, or at most 3. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the pH of thehybridization buffer may range from about 4 to about 8. Those of skillin the art will recognize that the pH of the hybridization buffer mayhave any value within this range, e.g., about pH 7.8. In some cases, thepH range is about 3 to about 10. In some embodiments, the disclosedhybridization buffer formulations may include adjustment of pH over therange of about pH 3 to pH 10, with a preferred buffer range of 5-9.

In some embodiments, the high efficiency hybridization buffer includesan additive (e.g., polar aprotic solvent) for controlling meltingtemperature of nucleic acid can vary depending on other agents used inthe compositions. The amount of the additive for controlling meltingtemperature of the nucleic acid is about or more than about 1%, 2%, 3%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or higher, by volumebased on the total volume of the formulation. In some cases, the amountof the additive for controlling melting temperature of the nucleic acidis greater than about 2% by volume based on the total volume of theformulation. In some cases, the amount of the additive for controllingmelting temperature of the nucleic acid is greater than 5% by volumebased on the total volume of the formulation. In some cases, the amountof the additive for controlling melting temperature of the nucleic acidis lower than about 3%, 5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%,50%, 60%, 70%, 80%, 90%, or higher, by volume based on the total volumeof the formulation. In some embodiments, the amount of the additive forcontrolling melting temperature of the nucleic acid is in the range ofabout 1% to 40%, 1% to 35%, 2% to 50%, 2% to 40%, 2% to 35%, 2% to 30%,2% to 25%, 2% to 20%, 2% to 10%, 5% to 50%, 5% to 40%, 5% to 35%, 5% to30%, 5% to 25%, 5% to 20%, by volume based on the total volume of theformulation. In some embodiments, the amount of the additive forcontrolling melting temperature of the nucleic acid is in the range ofabout 2% to 20% by volume based on the total volume of the formulation.In some cases, the amount of the additive for controlling meltingtemperature of the nucleic acid is in the range of about 5% to 10% byvolume based on the total volume of the formulation.

In some embodiments, the high efficiency hybridization bufferformulations may include the addition of an additive that alters nucleicacid duplex melting temperature. Examples of a suitable additive thatmay be used to alter nucleic acid melting temperature include, but arenot limited to, formamide. In some embodiments, the percentage by volumeof a melting temperature additive included in the high efficiencyhybridization buffer formulation may range from about 1% to about 50%.In some embodiments, the percentage by volume of a melting temperatureadditive may be at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, or at least 50%. In some embodiments, the percentage byvolume of a melting temperature additive may be at most 50%, at most45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, atmost 15%, at most 10%, at most 5%, or at most 1%. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of a melting temperature additive may range from about 10% toabout 25%. Those of skill in the art will recognize that the percentageby volume of a melting temperature additive may have any value withinthis range, e.g., about 22.5%.

In some embodiments, the hybridization buffer described herein includesan additive that impacts DNA hydration: In some embodiments, thedisclosed hybridization buffer formulations may include the addition ofan additive that impacts nucleic acid hydration. Examples include, butare not limited to, betaine, urea, glycine betaine, or any combinationthereof. In some embodiments, the percentage by volume of a hydrationadditive included in the hybridization buffer formulation may range fromabout 1% to about 50%. In some embodiments, the percentage by volume ofa hydration additive may be at least 1%, at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, or at least 50%. In some embodiments, thepercentage by volume of a hydration additive may be at most 50%, at most45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, atmost 15%, at most 10%, at most 5%, or at most 1%. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, the percentage byvolume of a hydration additive may range from about 1% to about 30%.Those of skill in the art will recognize that the percentage by volumeof a melting temperature additive may have any value within this range,e.g., about 6.5%.

Compositions and methods for preparing and using the high efficiencyhybridization buffers, are described in U.S. Ser. No. 16/543,351, filedon Aug. 16, 2019, the contents of which is hereby expressly incorporatedby reference in its entirety.

Air Bubble Fluidics

The present disclosure provides systems and methods for handlingfluidics that can be employed to conduct any of the pairwise sequencingmethods described herein, including any of the in-solution or on-supportrolling circle amplification reactions and/or any of the sequencingreactions. Pairwise sequencing workflows that are conducted on a flowcell in a massively parallel manner require numerous reagent changes toconduct different biochemical reactions throughout the workflow. Afluidic dispensing system can be used to deliver the reagents to theflow cell. The fluidic dispensing system needs to handle very smallfluid volumes and reduce mixing of different reagents that aresequentially flowed onto the flowcell. In some embodiments, air bubblescan be introduced into the fluidics system to separate differentreagents flowing onto the flow cell and reduce mixing.

The system comprises a flow cell fluidically connected to a reagentreservoir and a syringe pump. The flow cell includes an inlet on theupstream side and an outlet on the downstream side. The reagentreservoir is connected to the inlet of the flow cell and the syringepump is connected to the outlet of the flow cell, in a manner that formsa fluid flow path from the reagent reservoir, through the flow cell, andtowards the syringe pump. The system further includes an air bubbleinjector fluidically connected to the fluid flow path and configured toinject air bubbles between different reagents flowing through the fluidpath. Fluidic lines can be used to fluidically connect the reagentreservoir, bubble injector, flow cell and syringe pump.

The system can be used to flow very small volumes of different reagentsin the fluid flow path and reduce mixing of the different reagents. Forexample, a first fluid from the reagent reservoir enters the flow pathvia a fluidic line that connects the reagent reservoir and the inlet ofthe flow cell. An air bubble is injected into the flow path behind thefirst reagent in the fluidic line. A second fluid from the reagentreservoir enters the flow path via the fluidic line that connects thereagent reservoir and the inlet of the flow cell and the second reagentis behind the air bubble. The air bubble prevents mixing of the firstand second reagents in the fluidic line before the reagents reach theflow cell. Subsequent air bubbles and reagents can be flowed through theflow cell. The system further comprises an air bubble aspirator thatremoves the air bubble between the first and second reagents so that thefirst and second reagents are flowed sequentially onto the flow cellwith no air bubble. The air bubble aspirator is configured to enableintroduction of air bubbles between different reagents in the fluidicline before entry onto the flow cell so that air bubbles do not flowonto the flow cell. The syringe pump is configured to pull the first andsecond fluids and the air bubble along the flow path from the reagentreservoir and towards the outlet of the flow cell. In this manner,different reagents can be flowed sequentially onto the flow cell with noair bubbles.

The system includes at least one reagent reservoir each reservoir havinga plurality of separate compartments, and each compartment holding areagent for conducting a biochemical or biological reaction. Thereservoir(s) contain reagents for conducting nucleic acid amplificationand/or nucleic acid sequencing reactions on the flowcell. Eachcompartment can hold a different reagent in fluid form for conducting anucleic acid amplification reaction, a nucleic acid sequencing reaction,or washing reagent. For example, a first reservoir compartment can holda plurality of amplification polymerase, a plurality of amplificationprimers, a mixture of four different nucleotide triphosphates or analogsthereof (e.g., dATP, dGTP, dCTP or dTTP) and a nucleotide comprising ascissile moiety. A second reservoir compartment can hold a plurality ofsequencing polymerases, a plurality of soluble sequencing primers, and amixture of nucleotide triphosphates or analogs thereof (e.g., dATP,dGTP, dCTP or dTTP). A third reservoir compartment can hold a pluralityof sequencing polymerases, a plurality of soluble sequencing primers,and a mixture of multivalent molecules where individual multivalentmolecules comprises nucleotide arms having nucleotide units of dATP,dGTP, dCTP or dTTP. A fourth reservoir compartment can hold a pluralityof linear or circular library molecules. A fifth reservoir compartmentcan hold a wash buffer.

The system includes at least one flow cell having at least one channel.In some embodiments, the flow cell contains 1, 2, 3, 4, or morechannels. In some embodiments, the system includes two or more flowcells. Each flow cell can be configured with a plurality of channelsarranged in parallel to each other. Each channel has an inlet on theupstream side and an outlet on the downstream side. The flow cell(s)is/are mounted in the fluidics system so that the inlet side of eachchannel is/are fluidically connected to the reagent reservoir, and theoutlet side of each channel is/are fluidically connected to the syringepump.

The flow cell channels have a surface that can be coated with at leastone hydrophilic polymer layer, or at least one functionalized polymercoating. The channel surface can include at least one surface captureprimer immobilized on or embedded in the coating. The channel surfacecan include at least 1000 immobilized surface capture primers per mm².The immobilized surface capture primers on the surface of the channelcan be in fluid communication with each other during fluid flow throughthe channel to permit essentially simultaneously reaction with thereagents in a massively parallel manner. The immobilized surface captureprimers can hybridize to linear or circular nucleic acid librarymolecules each having a sequence of interest. The hybridized linear orcircular nucleic molecules can be subjected to an amplification reaction(e.g., a rolling circle amplification reaction) in the channel using thefluidic system to generate a plurality of concatemers. The plurality ofconcatemers in the channel can be subjected to successive sequencingreactions using the fluidic system.

EXAMPLES

The following examples are meant to be illustrative and can be used tofurther understand embodiments of the present disclosure and should notbe construed as limiting the scope of the present teachings in any way.

Example 1: On-Support Rolling Circle Amplification

On-Support Rolling Circle Amplification:

A support having a low non-specific binding coating with a plurality ofone type of surface primers (e.g., first surface primers) immobilizedthereon was prepared. The first surface primers included an extendible3′OH end and lacked any nucleotide having a scissile moiety (e.g.,lacked uracil, 8oxoG and deoxyinosine). Optionally, the coating alsoincluded a plurality of a second type of surface primers (e.g., secondsurface primers) which lacked any nucleotide having a scissile moietyand included a non-extendible 3′ phosphate group.

The circular library molecules included a sequence of interest (e.g.,insert size ranged from 150-400 bases) and a primer binding sequence foreach of the following: first surface primer; forward sequencing primer;and reverse sequencing primer. The circular library molecules optionallyincluded a primer binding sequence for a second surface primer.

A preparation of a single stranded circular nucleic acid library wasdistributed onto the support and incubated under a condition suitablefor hybridizing the circular library molecules to the first surfaceprimers to form library-primer duplexes. For example, the hybridizationwas conducted at 70° C. for about 3 minutes, and at 55° C. for 3minutes, and at 37° C. for 3 minutes, and at room temperature for 3minutes. The support was washed 3 times with a buffer containing Tris(pH 8), NaCl, EDTA and Tween-20.

A two-stage rolling circle amplification reaction was conducted. In thefirst stage, the library-primer duplexes were contacted with a stranddisplacing polymerase (e.g., phi29, amplifying polymerase) in thepresence of a binding buffer that lacked magnesium and nucleotides undera condition suitable for the polymerase to bind the library-primerduplexes to form complexed polymerases but inhibit polymerase-catalyzednucleotide incorporation. For example, the polymerase was incubated withthe library-primer duplexes at room temperature for about 15 minutes.The binding buffer was removed. In the second stage, the rolling circleamplification reaction (primer extension) was initiated by adding anextension buffer that included MgCl₂ a mixture of nucleotides (e.g.,about 1 mM each of dATP, dGTP, dCTP and dTTP) and dUTP (e.g., 1 μM or 10μM dUTP). The rolling circle amplification reaction contained varyingamounts of dUTP (e.g., about 1%, about 2.5%, about 5%, about 10% orabout 15%). Optionally, a plurality of condenser oligonucleotides wereadded (e.g., 25-200 nM). The rolling circle reaction was incubated at45° C. (isothermal condition) for about 60 minutes (or less than 60minutes) to generate a plurality of immobilized concatemers. Thereaction was cooled to room temperature and washed several times.

Read 1 Sequencing:

A two-stage sequencing reaction was conducted. The first-stagesequencing reaction was conducted by hybridizing a plurality of asoluble forward sequencing primers (e.g., 5′ exonuclease-resistantprimers) to the immobilized concatemers to form primer-concatemerduplexes. A plurality of a first sequencing polymerase was added to theduplexes and incubated under a condition suitable to bind the sequencingpolymerase to the duplexes to form complexed polymerases. A mixture offluorescently labeled multivalent molecules (e.g., about 40-100 nM) wasadded to the complexed polymerases in the presence of a buffer thatincluded a non-catalytic cation (e.g., strontium or barium) andincubated under conditions suitable to bind complementary nucleotideunits of the multivalent molecules to the complexed polymerases withoutpolymerase-catalyzed incorporation of the nucleotide units. Thecomplexed polymerases were washed. An image was obtained of thefluorescently labeled multivalent molecules that remined bound to thecomplexed polymerases. The first sequencing polymerases and multivalentmolecules were removed, while retaining the sequencing primershybridized to the concatemers (retained duplexes), by washing with abuffer comprising a detergent.

The second-stage sequencing reaction was conducted by contacting theretained duplexes with a plurality of second sequencing polymerases toform complexed polymerases. A mixture of fluorescently labelednucleotide analogs (e.g., 3′O-methylazido nucleotides) (e.g., about 1-5μM) was added to the complexed polymerases in the presence of a bufferthat included a catalytic cation (e.g., magnesium) and incubated underconditions suitable to bind complementary nucleotides to the complexedpolymerases and promote polymerase-catalyzed incorporation of thenucleotides to generate a nascent extended sequencing primer. Thecomplexed polymerases were washed. An image was obtained of theincorporated fluorescently labeled nucleotide analogs as a part of thecomplexed polymerases. The incorporated fluorescently labeled nucleotideanalogs were reacted with a cleaving reagent that removes the 3′O-methylazido group and generates an extendible 3′OH group. The secondsequencing polymerases were removed, while retaining the nascentextended sequencing primers hybridized to the concatemers (retainedduplexes), by washing with a buffer comprising a detergent. Recurringsequencing reactions were conducted by performing multiple cycles offirst-stage and second-stage sequencing reactions to generate extendedforward sequencing primer strands. FIG. 118 (left) shows a comparison oferror rates determined for the concatemers generated with varyingamounts of dUTP (e.g., about 1%, about 2.5%, about 5%, about 10% orabout 15%). FIG. 118 (right) shows a comparison of phasing ratesdetermined for the concatemers generated with varying amounts of dUTP(e.g., about 1%, about 2.5%, about 5%, about 10% or about 15%).

Replacing the Extended Forward Sequencing Primer Strands:

The extended forward sequencing primer strands were contacted with aplurality of strand displacing polymerases (e.g., Phi29 polymerase) anda mixture of nucleotides in the absence of newly added soluble primers(e.g., no soluble sequencing primers and no soluble amplificationprimers). The mixture of nucleotides included dATP, dGTP, dCTP and dTTP.Optionally, compaction oligonucleotides were added (e.g., about 100 μM).The reaction was incubated at 45° C. for about 30 minutes to generate aplurality of forward extension strands that are hybridized to theimmobilized concatemer molecules. The support was washed several timeswith a wash buffer comprising Tris-HCl, NaCl, EDTA and Tween-20.

Generating Abasic Sites and Gaps:

The plurality of forward extension strands were contacted withThermolabile USER (II) enzyme (0.02 U/uL) (e.g., from New EnglandBiolabs, catalog #M5508S), and T7 exonuclease (02 U/uL) (e.g., from NewEngland Biolabs, catalog #M0263S), and 1× CutSmart buffer (e.g., fromNew England Biolabs, catalog #B7204S), and incubated at 37° C. for about15 minutes, and then at 25° C. for about 15 minutes to generate aplurality of abasic sites in the immobilized concatemer molecules and togenerate gaps at the abasic sites. The forward extension strands wereretained as intact molecules. The support was washed several times witha wash buffer comprising Tris-HCl, NaCl, EDTA and Tween-20 to remove thegap-containing concatemer molecules.

Read 2 Sequencing:

A two-stage sequencing reaction was conducted as described in ‘Read 1sequencing’ described above, except that the first-stage sequencingreaction was conducted by hybridizing a plurality of a soluble reversesequencing primers (e.g., 5′ exonuclease-resistant primers) to theretained forward extension strands to form primer-extension strandduplexes.

Example 2: In-Solution Initiated Rolling Circle Amplification

In-Solution Initiated Rolling Circle Amplification:

A support having a low non-specific binding coating with a plurality ofone type of surface primers (e.g., first surface primers) immobilizedthereon was prepared. The first surface primers included an extendible3′OH end and lacked any nucleotide having a scissile moiety (e.g.,lacked uracil, 8oxoG and deoxyinosine). Optionally, the coating alsoincluded a plurality of a second type of surface primers (e.g., secondsurface primers) which lacked any nucleotide having a scissile moietyand included a non-extendible 3′ phosphate group.

The circular library molecules included a sequence of interest (e.g.,insert size ranged from 150-400 bases) and a primer binding sequence foreach of the following: a soluble amplification primer; first surfaceprimer; forward sequencing primer; and reverse sequencing primer. Thecircular library molecules optionally included a primer binding sequencefor a second surface primer.

The circular library molecules (at 1-8 nM) were hybridized with solubleamplification primers (at 2-80 nM)(e.g., about 2-10 equivalents) in abuffer containing 10 mM ACES pH 7.4, 10 μM dNTPs (total), 1 mM strontiumacetate, 0.01% Tween-20, (and optionally mM ammonium sulfate). Thehybridization mixture was heated to 85° C. and cooled to 40° C. with 5°C. steps, 30 second dwell time at each step, and 0.2° C./second ramprate between steps, for a total of 10 minutes. The mixture was incubatedat a temperature that was about 1-10° C. below the Tm of the primer, forabout 10 minutes.

To the hybridization mixture described above, a trappednucleotide-polymerase mixture was prepared by adding DTT to a finalconcentration of 1-50 mM, Phi29 polymerase at 2-10 equivalents withrespect to the soluble amplification primer, and a 5× trap buffer sothat the final concentration of the ACES, dNTPs, strontium acetate andTween-20 was the same as in the hybridization mixture. The trappednucleotide-polymerase mixture was incubated at room temperature, ordifferent temperatures up to 35° C., for 15 minutes.

A nucleotide polymerization reaction mixture was prepared by dilutingthe trapped nucleotide-polymerase mixture (about 40-1000× dilution) withextension buffer so the final concentration included 50 mM ACES pH 74,100 mM potassium acetate, 10 mM magnesium sulfate, 2 mM dNTPs, 10 mMDTT, 0.01% Tween-20, 50 mM ammonium sulfate, and concentration of thecircular library molecules was 5-100 pM.

The nucleotide polymerization reaction mixture was distributed onto asupport immobilized with two different types of surface capture primers,and incubated at 30-45° C. for 5 minutes (but time ranges up to 2 hourswere also tested) for a rolling circle amplification reaction.Alternatively, the nucleotide polymerization reaction mixture wasincubated at 30-45° C. for 30 minutes and then distributed onto thesupport, and the rolling circle amplification reaction continued on thesurface for 5 minutes or up to 2 hours.

The support was washed with a wash buffer containing 50 mM Tris-HCl pH8, 750 mM NaCl, 0.1 mM EDTA, and 0.02% Tween-20. The reaction was cooledto room temperature and washed several times.

Read 1 Sequencing:

A two-stage sequencing reaction was conducted as described above inExample 1.

Replacing the Extended Forward Sequencing Primer Strands

The extended forward sequencing primer strands were replaced withforward extension strands as described above in Example 1.

Generating Abasic Sites and Gaps

Abasic sites and gaps were generated in the forward extension strands asdescribed above in Example 1.

Read 2 Sequencing:

A two-stage sequencing reaction was conducted as described above inExample 1.

What is claimed:
 1. A method for pairwise sequencing, comprising: a)providing a plurality of immobilized single stranded nucleic acidconcatemer template molecules, wherein individual concatemer templatemolecules in the plurality comprise at least one nucleotide having ascissile moiety that can be cleaved to generate an abasic site in theconcatemer template molecule, wherein each individual concatemertemplate molecule in the plurality is immobilized to a first surfaceprimer that is immobilized to a support, and wherein the immobilizedfirst surface primers lack nucleotides having a scissile moiety; b)sequencing the plurality of immobilized concatemer template molecules,thereby generating a plurality of extended forward sequencing primerstrands, wherein individual immobilized concatemer template moleculeshave two or more extended forward sequencing primer strands hybridizedthereon; c) retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized concatemer template molecules byconducting a primer extension reaction; d) removing the retainedimmobilized concatemer template molecules by generating abasic sites inthe immobilized concatemer template molecules at the nucleotide(s)having the scissile moiety and generating gaps at the abasic sites togenerate a plurality of gap-containing single stranded nucleic acidconcatemer template molecules while retaining the plurality of forwardextension strands and retaining the plurality of immobilized surfaceprimers; and e) sequencing the plurality of retained forward extensionstrands, thereby generating a plurality of extended reverse sequencingprimer strands, wherein individual retained forward extension strandshave two or more extended reverse sequencing primer strands hybridizedthereon.
 2. The method of claim 1, wherein each individual concatemertemplate molecule in the plurality is covalently joined to animmobilized first surface primer.
 3. The method of claim 1, wherein eachindividual concatemer template molecule in the plurality is hybridizedto an immobilized first surface primer.
 4. The method of claim 1,wherein individual immobilized concatemer template molecules in theplurality comprise two or more copies of a sequence of interest and anyone or any combination of two or more of: (i) two or more copies of auniversal binding sequence for a soluble forward sequencing primer, (ii)two or more copies of a universal binding sequence for a soluble reversesequencing primer, (iii) two or more copies of a universal bindingsequence for an immobilized first surface primer, (iv) two or morecopies of a universal binding sequence for an immobilized second surfaceprimer, (v) two or more copies of a universal binding sequence for afirst soluble amplification primer, (vi) two or more copies of auniversal binding sequence for a second soluble amplification primer,(vii) two or more copies of a universal binding sequence for a solublecompaction oligonucleotide, (viii) two or more copies of a samplebarcode sequence and/or (ix) two or more copies of a unique molecularindex sequence.
 5. The method of claim 1, wherein the sequencing of step(b) comprises hybridizing a plurality of soluble forward sequencingprimers to the plurality of immobilized concatemer template moleculesand conducting one or more sequencing reactions.
 6. The method of claim1, wherein the sequencing of step (e) comprises hybridizing a pluralityof soluble reverse sequencing primers to the plurality of retainedforward extension strands and conducting one or more sequencingreactions.
 7. The method of claim 4, wherein the support furthercomprises a plurality of immobilized second surface primers that lack anucleotide having a scissile moiety.
 8. The method of claim 7, whereinat least one copy of the universal binding sequence for the immobilizedsecond surface primer in the individual concatemer template molecules ishybridized to an immobilized second surface primer.
 9. The method ofclaim 7, wherein the plurality of immobilized second surface primershave 3′ OH extendible ends.
 10. The method of claim 7, wherein theplurality of immobilized second surface primers have 3′ non-extendibleends.
 11. The method of claim 10, wherein the 3′ non-extendible endcomprises a phosphate group, a dideoxycytidine group, an inverted dT, oran amino group.
 12. The method of claim 1, comprising at (a): i)providing a support having a plurality of a first surface primersimmobilized thereon, wherein the first surface primers have 3′extendible ends; and ii) generating a plurality of immobilized singlestranded nucleic acid concatemer template molecules by hybridizing aplurality of single-stranded circular nucleic acid library molecules tothe plurality of immobilized first surface primers and conducting arolling circle amplification reaction with a plurality of stranddisplacing polymerases, and a plurality of nucleotides which includedATP, dCTP, dGTP, dTTP and nucleotides having a scissile moiety that canbe cleaved to generate an abasic site, thereby generating a plurality ofimmobilized single stranded nucleic acid concatemer template molecules,wherein one or more individual immobilized single stranded nucleic acidconcatemer template molecules within the plurality have at least onenucleotide with a scissile moiety, and wherein individual singlestranded nucleic acid concatemer template molecules are covalentlyjoined to immobilized first surface primers.
 13. The method of claim 12,wherein individual single-stranded circular nucleic acid librarymolecules in the plurality comprise a sequence of interest and any oneor any combination of two or more of: (i) a universal binding sequencefor a soluble forward sequencing primer, (ii) a universal bindingsequence for a soluble reverse sequencing primer, (iii) a universalbinding sequence for an immobilized first surface primer, (iv) auniversal binding sequence for an immobilized second surface primer, (v)a universal binding sequence for a first soluble amplification primer,(vi) a universal binding sequence for a second soluble amplificationprimer, (vii) a universal binding sequence for a soluble compactionoligonucleotide, (viii) a sample barcode sequence and/or (ix) a uniquemolecular index sequence.
 14. The method of claim 1, comprising prior to(a): i) contacting in-solution a plurality of single-stranded circularnucleic acid library molecules with a plurality of first solubleamplification primers, a plurality of a strand displacing polymerases,and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP andnucleotides having a scissile moiety that can be cleaved to generate anabasic site, under conditions suitable to form a plurality oflibrary-primer duplexes and conduct a rolling circle amplificationreaction, thereby generating a plurality of single stranded nucleic acidconcatemers, wherein one or more individual single stranded nucleic acidconcatemers within the plurality have at least one nucleotide with ascissile moiety; ii) distributing the rolling circle amplificationreaction onto a support having a plurality of the first surface primersimmobilized thereon, under conditions suitable for hybridizing one ormore portions of individual single stranded concatemers to one or moreimmobilized first surface primers, wherein the first surface primerslack a nucleotide having a scissile moiety; and iii) continuing therolling circle amplification reaction on the support to generate aplurality of immobilized single stranded concatemer template molecules.15. The method of claim 14, wherein individual single-stranded circularnucleic acid library molecules in the plurality comprise a sequence ofinterest and any one or any combination of two or more of: (i) auniversal binding sequence for a soluble forward sequencing primer, (ii)a universal binding sequence for a soluble reverse sequencing primer,(iii) a universal binding sequence for an immobilized first surfaceprimer, (iv) a universal binding sequence for an immobilized secondsurface primer, (v) a universal binding sequence for a first solubleamplification primer, (vi) a universal binding sequence for a secondsoluble amplification primer, (vii) a universal binding sequence for asoluble compaction oligonucleotide, (viii) a sample barcode sequenceand/or (ix) a unique molecular index sequence.
 16. A method for pairwisesequencing, comprising: a) providing a support having a plurality of afirst surface primers immobilized thereon, wherein individualimmobilized first surface primers in the plurality comprise a firstportion (SP1-A) and a second portion (SP1-B), and wherein individualfirst surface primers comprise a 3′ extendible end and lack a nucleotidehaving a scissile moiety that can be cleaved to generate an abasic sitein the first surface primer; b) contacting the plurality of the firstsurface primers with a plurality of single stranded linear nucleic acidlibrary molecules, wherein individual library molecules have a universalsequence (SP1-A′) at the 5′ end that binds to the first portion of theimmobilized first surface primers and a universal sequence (SP1-B′) atthe 3′ end that binds to the second portion of the immobilized firstsurface primers, and wherein the contacting is conducted underconditions suitable for hybridizing individual library molecules withthe immobilized first surface primers to form circularized librarymolecules, wherein individual circularized library molecules have a gapor nick between the 5′ and 3′ ends of the circularized library molecule;c) enzymatically closing the gap or nick, thereby forming covalentlyclosed circular molecules that are hybridized to the plurality ofimmobilized first surface primers; d) generating a plurality ofimmobilized single stranded nucleic acid concatemer template moleculesby conducting a rolling circle amplification reaction with a pluralityof a strand displacing polymerases, and a plurality of nucleotides whichinclude dATP, dCTP, dGTP, dTTP and nucleotides having a scissile moietythat can be cleaved to generate an abasic site, thereby generating aplurality of immobilized single stranded nucleic acid concatemertemplate molecules, wherein one or more individual concatemer templatemolecules within the plurality have at least one nucleotide with ascissile moiety, and wherein individual concatemer template moleculesare covalently joined to immobilized first surface primers; e)sequencing the plurality of immobilized concatemer template molecules,thereby generating a plurality of extended forward sequencing primerstrands, wherein individual immobilized concatemer template moleculeshave two or more extended forward sequencing primer strands hybridizedthereon; f) retaining the plurality of immobilized concatemer templatemolecules and replacing the plurality of extended forward sequencingprimer strands with a plurality of forward extension strands that arehybridized to the retained immobilized concatemer template molecules byconducting a primer extension reaction; g) removing the retainedimmobilized concatemer template molecules by generating abasic sites inthe immobilized single stranded concatemer template molecules at thenucleotide(s) having the scissile moiety and generating gaps at theabasic sites to generate a plurality of gap-containing single strandednucleic acid concatemer template molecules while retaining the pluralityof forward extension strands and retaining the plurality of immobilizedfirst surface primers; and h) sequencing the plurality of retainedforward extension strands, thereby generating a plurality of extendedreverse sequencing primer strands, wherein individual forward extensionstrands have two or more extended reverse sequencing primer strandshybridized thereon.
 17. A method for pairwise sequencing, comprising: a)providing a plurality of immobilized single stranded nucleic acidconcatemer template molecules, wherein individual concatemer templatemolecules lack a scissile moiety that can be cleaved to generate anabasic site in the concatemer template molecule, wherein individualconcatemer template molecules in the plurality are immobilized to afirst surface primer that is immobilized to a support, and wherein theimmobilized first surface primer lacks a nucleotide having a scissilemoiety; b) sequencing the plurality of immobilized concatemer templatemolecules, thereby generating a plurality of extended forward sequencingprimer strands, wherein individual immobilized concatemer templatemolecules have two or more extended forward sequencing primer strandshybridized thereon; c) retaining the plurality of immobilized concatemertemplate molecules and replacing the plurality of extended forwardsequencing primer strands with a plurality of forward extension strandsby conducting a primer extension reaction with a plurality of solubleamplification primers and a plurality of strand-displacing polymerasesto generate a plurality of forward extension strands and a plurality ofpartially displaced forward extension strands that are hybridized to theimmobilized concatemer template molecules to form a plurality ofimmobilized amplicons, and wherein the primer extension reactiongenerates a plurality of detached forward extension strands; and d)sequencing the plurality of immobilized partially displaced forwardextension strands, thereby generating a first plurality of extendedreverse sequencing primer strands, and sequencing the plurality ofimmobilized detached forward extension strands, thereby generating asecond plurality of extended reverse sequencing primer strands, whereinindividual immobilized partially displaced forward extension strandshave two or more extended reverse sequencing primer strands hybridizedthereon, and wherein individual immobilized detached forward extensionstrands have two or more extended reverse sequencing primer strandshybridized thereon.
 18. A method for pairwise sequencing, comprising: a)providing a plurality of immobilized single stranded nucleic acidconcatemer template molecules, wherein individual concatemer templatemolecules in the plurality comprise at least one nucleotide having ascissile moiety that can be cleaved to generate an abasic site in theconcatemer template molecule, wherein individual concatemer templatemolecules in the plurality are immobilized to first surface primers thatare immobilized to a support, wherein individual immobilized firstsurface primers include a nucleotide having a scissile moiety, whereinthe support further comprises a plurality of immobilized second surfaceprimers which lack a nucleotide having a scissile moiety and have anextendible terminal 3′OH group, and wherein the immobilized concatemertemplate molecules comprise two or more copies of a universal bindingsequence for the immobilized second surface primers; b) sequencing theplurality of immobilized concatemer template molecules with a pluralityof soluble forward sequencing primers thereby generating a plurality ofextended forward sequencing primer strands, wherein individualimmobilized concatemer template molecules have two or more extendedforward sequencing primer strands hybridized thereon; c) removing theextended forward sequencing primer strands and retaining the immobilizedconcatemer template molecules; d) generating a first plurality ofimmobilized forward extension strands by hybridizing at least oneportion of individual immobilized concatemer template molecules to asecond surface primer and conducting a primer extension reaction fromthe second surface primers that are hybridized to a portion of theimmobilized concatemer template molecules to generate a plurality offorward extension strands having sequences that are complementary to atleast a portion of the immobilized concatemer template molecules and arecovalently joined to an immobilized second surface primer; e) contactingthe plurality of immobilized concatemer template molecules and theplurality of immobilized forward extension strands with a relaxingsolution which comprises at least one chaotropic agent; f) dissociatingthe at least one portion of the immobilized concatemer templatemolecules from the immobilized second surface primers and retaining theimmobilized forward extension strands, and re-hybridizing at least oneportion of the individual immobilized concatemer template molecules toan immobilized second surface primer that is not covalently joined to aforward extension strand, wherein the dissociating and re-associatingcomprises a temperature ramp-up, a temperature plateau, and temperatureramp-down, and washing the relaxing solution from the support; g)contacting the re-hybridized immobilized concatemer template moleculeswith an amplification solution and conducting a primer extensionreaction from the second surface primers that are re-hybridized to aportion of the immobilized concatemer template molecules to generate aplurality of newly synthesized forward extension strands having asequence that is complementary to at least a portion of the immobilizedconcatemer template molecules and are covalently joined to immobilizedsecond surface primers; h) repeating steps (e)-(g) at least once; i)removing the immobilized concatemer template molecules by generatingabasic sites in the immobilized concatemer template molecules and theimmobilized first surface primers at the nucleotide(s) having thescissile moiety and generating gaps at the abasic sites, therebygenerating a plurality of gap-containing nucleic acid molecules whileretaining the plurality of immobilized forward extension strands andretaining the plurality of immobilized second surface primers; and j)sequencing the plurality of retained immobilized forward extensionstrands with a plurality of soluble reverse sequencing primers, therebygenerating a plurality of extended reverse sequencing primer strands.19. The method of claim 1, wherein the support comprises a planarsubstrate which comprises glass, fused-silica, silicon, a polymer, orany combination thereof.
 20. The method of claim 1, wherein the supportcomprises at least one hydrophilic polymer coating having a watercontact angle of no more than 45 degrees, and wherein at least one ofthe hydrophilic polymer coatings comprises a branched hydrophilicpolymer having at least 4 branches.
 21. The method of claim 1, whereinthe 5′ ends of the plurality of first surface primers are immobilized tothe support or immobilized to a coating on the support.
 22. The methodof claim 1, wherein the at least one nucleotide having a scissile moietycomprises uridine, 8-oxo-7,8-dihydrogunine, or deoxyinosine.
 23. Themethod of claim 1, wherein the nucleotides with a scissile moiety arelocated at randomly distributed positions in individual immobilizedconcatemer template molecules in the plurality.
 24. The method of claim1, wherein 0.01-30% of the thymidine nucleotides in the individualimmobilized concatemer template molecules are replaced with uridine. 25.The method of claim 1, wherein the forward sequencing of step (b)comprises: a) contacting a plurality of sequencing polymerases with (i)a plurality of immobilized concatemer template molecules and (ii) aplurality of the soluble forward sequencing primers, wherein thecontacting is conducted under conditions suitable to form a plurality ofcomplexed polymerases comprising a sequencing polymerase bound to anucleic acid duplex wherein the nucleic acid duplex comprises animmobilized concatemer template molecule hybridized to a soluble forwardsequencing primer; b) contacting the plurality of complexed polymeraseswith a plurality of nucleotides under conditions suitable for binding atleast one nucleotide to the complexed polymerases, wherein the pluralityof nucleotides comprises at least one nucleotide analog labeled with afluorophore and having a removable chain terminating moiety at the sugar3′ position; c) incorporating at least one nucleotide into the 3′ endsof the hybridized forward sequencing primers, thereby generating aplurality of nascent extended forward sequencing primers; and d)detecting the incorporated nucleotide and identifying the nucleo-base ofthe incorporated nucleotide.
 26. The method of claim 1, wherein thereverse sequencing of step (e) comprises: a) contacting a plurality ofsequencing polymerases with (i) the plurality of retained forwardextension strands and (ii) a plurality of the soluble reverse sequencingprimers, wherein the contacting is conducted under conditions suitableto form a plurality of complexed polymerases comprising a sequencingpolymerase bound to a nucleic acid duplex wherein the nucleic acidduplex comprises a retained forward extension strand hybridized to asoluble reverse sequencing primer; b) contacting the plurality ofcomplexed polymerases with a plurality of nucleotides under conditionssuitable for binding at least one nucleotide to the complexedpolymerases, wherein the plurality of nucleotides comprises at least onenucleotide analog labeled with a fluorophore and having a removablechain terminating moiety at the sugar 3′ position; c) incorporating atleast one nucleotide into the 3′ ends of the hybridized reversesequencing primers thereby generating a plurality of nascent extendedreverse sequencing primers; and d) detecting the incorporated nucleotideand identifying the nucleo-base of the incorporated nucleotide.
 27. Themethod of claim 1, wherein the forward sequencing of step (b) and thereverse sequencing of step (e) comprise: 1) conducting a sequencingreaction at a position on the concatemer template molecules usingmultivalent molecules which bind but do not incorporate; 2) conducting asequencing reaction at the same position on the concatemer templatemolecules using nucleotides with incorporation; and 3) repeating steps(a) and (b) at the next position on the concatemer template molecules.28. The method of claim 1, wherein the forward sequencing of step (b)and the reverse sequencing of step (e) comprise: a) contacting aplurality of a first polymerases with (i) a plurality of nucleic acidtemplate molecules and (ii) a plurality of soluble sequencing primers,wherein the contacting is conducted under conditions suitable to form aplurality of first complexed polymerases comprising a first sequencingpolymerase bound to nucleic acid duplexes, wherein the nucleic acidduplexes comprise the nucleic acid template molecule hybridized to thesequencing primer, wherein (1) the plurality of nucleic acid templatemolecules comprise a plurality of the immobilized concatemer templatemolecules and the plurality of soluble primers comprise a plurality ofthe soluble forward sequencing primers, or wherein (2) the plurality ofnucleic acid template molecules comprise a plurality of the retainedforward extension strands and the plurality of soluble sequencingprimers comprise a plurality of the soluble reverse sequencing primers;b) contacting the plurality of first complexed polymerases with aplurality of detectably labeled multivalent molecules, whereinindividual multivalent molecules comprise a core attached to multiplenucleotide arms, wherein each nucleotide arm is attached to a nucleotideunit, thereby forming a plurality of multivalent-complexed polymerases,under conditions suitable for binding complementary nucleotide units toat least two of the plurality of first complexed polymerases, therebyforming a plurality of multivalent-complexed polymerases, and whereinthe conditions inhibit incorporation of the complementary nucleotideunits into the sequencing primers of the nucleic acid duplexes; c)detecting the plurality of multivalent-complexed polymerases; and d)identifying the nucleo-base of the complementary nucleotide units thatare bound to the plurality of first complexed polymerases in theplurality of multivalent-complexed polymerases, thereby determining thesequence of the nucleic acid template.
 29. The method of claim 28,comprising forming at least one avidity complex in step (b), whereinforming the at least one avidity complex comprises: a) binding a firstsequencing primer, a first sequencing polymerase, and a firstmultivalent molecule to a first portion of a nucleic acid templatemolecule thereby forming a first binding complex, wherein a firstnucleotide unit of the first multivalent molecule binds to the firstsequencing polymerase; and b) binding a second sequencing primer, asecond sequencing polymerase, and the first multivalent molecule to asecond portion of the same nucleic acid template molecule therebyforming a second binding complex, wherein a second nucleotide unit ofthe second multivalent molecule binds to the second sequencingpolymerase, wherein the first and second binding complexes which includethe same multivalent molecule forms an avidity complex.
 30. The methodof claim 25, wherein individual nucleotides in the plurality ofnucleotides comprise an aromatic base, a five carbon sugar, and 1-10phosphate groups, and wherein the aromatic base of the nucleotidecomprises adenine, guanine, cytosine, thymine or uracil.
 31. The methodof claim 30, wherein at least one of the nucleotides in the plurality ofnucleotides comprises a fluorescently-labeled nucleotide.
 32. The methodof claim 25, wherein at least one of the nucleotides in the plurality ofnucleotides comprises a chain terminating moiety attached to a 3′-OHsugar position via a cleavable moiety, and wherein the chain terminatingmoiety comprises an alkyl group, alkenyl group, alkynyl group, allylgroup, aryl group, benzyl group, azide group, amine group, amide group,keto group, isocyanate group, phosphate group, thio group, disulfidegroup, carbonate group, urea group, or silyl group.
 33. The method ofclaim 27, wherein individual multivalent molecules in the plurality ofmultivalent molecules comprise (a) a core; and (b) a plurality ofnucleotide arms comprising (i) a core attachment moiety, (ii) a spacercomprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit,wherein the core is attached to the plurality of nucleotide arms viatheir core attachment moiety, wherein the spacer is attached to thelinker, and wherein the linker is attached to the nucleotide unit. 34.The method of claim 33, wherein: (i) the core comprises an avidin-typemoiety and the core attachment moiety comprises biotin; (ii) the linkercomprises an aliphatic chain having 2-6 subunits or an oligo ethyleneglycol chain having 2-6 subunits; (iii) the nucleotide unit comprises anaromatic base, a five carbon sugar and 1-10 phosphate groups; (iv) thelinker is attached to the nucleotide unit through the base; (v) thelinker further comprises an aromatic moiety; (vi) the plurality ofnucleotide arms attached to the core have the same type of a nucleotideunit, and wherein the type of nucleotide unit is selected from the groupconsisting of dATP, dGTP, dCTP, dTTP and dUTP; or (vii) the plurality ofmultivalent molecules are fluorescently-labeled multivalent molecules.35. The method of claim 34, wherein (i) the core of individualfluorescently-labeled multivalent molecules is attached to a fluorophorewhich corresponds to the type of nucleotide unit attached to thenucleotide arms; (ii) at least one of the nucleotide arms comprises alinker that is attached to a fluorophore which corresponds to the typeof nucleotide unit attached to the nucleotide arms; and/or (iii) atleast one of the nucleotide arms comprises a nucleotide unit that isattached to a fluorophore which corresponds to the type of nucleotideunit attached to the nucleotide arms.
 36. The method of claim 33,wherein at least one of the multivalent molecules in the plurality ofmultivalent molecules comprises nucleotide units having a chainterminating moiety attached to the 3′-OH sugar position via a cleavablemoiety, and wherein the chain terminating moiety comprises an alkylgroup, alkenyl group, alkynyl group, allyl group, aryl group, benzylgroup, azide group, amine group, amide group, keto group, isocyanategroup, phosphate group, thio group, disulfide group, carbonate group,urea group, or silyl group.
 37. The method of claim 1, wherein step (c)comprises: (i) contacting the plurality of extended forward sequencingprimer strands with a plurality of strand displacing polymerases and aplurality of nucleotides and in the absence of soluble amplificationprimers, under conditions suitable to conduct a strand displacing primerextension reaction using the plurality of extended forward sequencingprimer strands to initiate the primer extension reaction, therebygenerating forward extension strands that are covalently joined to theextended forward sequencing primer strands, wherein the forwardextension strands are hybridized to the immobilized concatemer templatemolecules.
 38. The method of claim 1, wherein step (c) comprisesremoving the plurality of extended forward sequencing primer strands by:(i) contacting the plurality of extended forward sequencing primerstrands with a 5′ to 3′ double-stranded DNA exonuclease; (ii) contactingthe plurality of extended forward sequencing primer strands with adenaturation reagent comprising any combination of formamide,acetonitrile, guanidinium chloride and/or a pH buffering agent; or (iii)contacting the plurality of extended forward sequencing primer strandswith 100% formamide.
 39. The method of claim 1, wherein step (c)comprises contacting the plurality of retained immobilized concatemermolecules with a second plurality of soluble forward sequencing primers,a plurality of nucleotides and a plurality of primer extensionpolymerases, under conditions suitable to hybridize the second pluralityof soluble forward sequencing primers to the plurality of retainedimmobilized concatemer template molecules and conducting apolymerase-catalyzed primer extension reaction, thereby generating aplurality of forward extension strands replacing the plurality ofextended forward sequencing primer strands, wherein the plurality ofnucleotides comprise dATP, dGTP, dCTP and dTTP but lacks dUTP, whereinin the plurality of primer extension polymerases is tolerant ofuridine-containing template strands, and wherein the soluble sequencingprimers hybridize with the forward sequencing primer binding sequence inthe retained immobilized concatemer molecules.
 40. The method of claim1, wherein the primer extension reaction at step (c) comprisescontacting the plurality of retained immobilized concatemer moleculeswith a plurality of soluble amplification primers, a plurality ofnucleotides and a plurality of primer extension polymerases, underconditions suitable to hybridize the plurality of soluble amplificationprimers to the plurality of retained immobilized concatemer templatemolecules and a conducting polymerase-catalyzed primer extensionreaction, thereby generating a plurality of forward extension strands,wherein the soluble amplification primers hybridize with the solubleamplification primer binding sequence in the retained immobilizedconcatemer molecules, wherein the plurality of nucleotides comprisedATP, dGTP, dCTP and dTTP but lacks dUTP, wherein in the plurality ofprimer extension polymerases is tolerant of uridine-containing templatestrands, and wherein the soluble sequencing primers hybridize with theforward sequencing primer binding sequence in the retained immobilizedconcatemer molecules.
 41. The method of claim 1, wherein at least one ofthe retained immobilized concatemer template molecules includes one ormore nucleotides having a scissile moiety, and wherein the scissilemoiety comprises uridine or 8-oxo-7,8-dihydroguanine, or deoxyinosine.42. The method of claim 1, wherein the retained immobilized concatemertemplate molecules comprise one or more uridines, and wherein thegenerating the abasic sites at the uridines comprises contacting theretained immobilized concatemer template molecules with uracil DNAglycosylase (UDG).
 43. The method of claim 12, wherein the rollingcircle amplification of step ii) comprises a plurality of compactionoligonucleotides and/or hexamine to generate immobilized concatemertemplate molecules having a more compact size and/or shape compared to arolling circle amplification reaction in the absence of compactionoligonucleotides and/or hexamine.
 44. The method of claim 14, whereinthe rolling circle amplification reaction of step i) comprises aplurality of compaction oligonucleotides and/or hexamine to generateconcatemer molecules having a more compact size and/or shape compared toa rolling circle amplification reaction in the absence of compactionoligonucleotides and/or hexamine.
 45. The method of claim 18, wherein atstep (a) the support comprises an excess of immobilized first and secondsurface primers compared to the number of immobilized concatemertemplate molecules.
 46. The method of claim 19, wherein the polymercomprises polystyrene (PS), macroporous polystyrene (MPPS),polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP),polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), or polyethyleneterephthalate (PET).